Making neurons from stem cells: Molecular mechanisms and spider silk substrates

The understanding of the function of the nervous system and the brain is one of the major intellectual challenges in life sciences. Neurological and psychiatric disorders are in addition major issues for the society, and new approaches are needed to learn more about the brain and to develop new treatments. The development of the mammalian brain is a highly regulated process that involves extra-and intracellular signaling to efficiently regulate gene expression in a precise spatial and temporal manner.The understanding of the differentiation mechanisms into neurons, glia and other cell types in thedeveloping forebrain however is still incomplete. Studies of embryonic telencephalic neural stem cells (NSCs)in vitromay increase the understanding of the molecular mechanisms of brain development, and aid in developing new protocols for defined differentiation of stem cells for clinical use. This thesisis aimed at investigating the mechanisms underlying bone morphogenetic protein(BMP4)-mediated differentiation of NSCs, and to explore the use of recombinant spider silk protein-basedmatrices in combination with signaling factors, especially BMP4, to generate functional neural cell circuitsin vitro. In the first study we discovered that BMP4 treatment of NSCs resulted in a dramatic increase in theexpression of the BMP4-inhibitor Noggin. BMP4 mediated non-neural differentiation intomesenchymal cells at low seeding densities, neuronal differentiation at high seeding densities, andastrocyte differentiation in any condition. As the Noggin levels increased linearly at higher densities, wehypothesized that the endogenous Noggin production predominantly mediated an inhibition ofmesenchymal differentiation. We further observed that BMP4 stimulation induced an AMPAresponsive neuron population at high seeding densities, and that this population was increased by co-stimulation of the signaling factor Wnt3a. By applying whole transcriptome sequencing, we aimed at elucidating the molecular mechanisms responsible for the increased neuronal differentiation by BMP4+Wnt3a. This approach,however revealed an unexpected increase in the expression of genes associated with inhibitory GABAergicneurons, and also functional the expression of the neurogenic bHLH factor Hes6. To apply these novel protocols for differentiation of NSCs into functional neurons, we introduced anovel way of culturing NSCs in substrates generated from recombinant spider silk protein (4RepCT).Spider silk protein is a promising biomaterial due to its biocompatibility, biodegradability, andpossibility to use in various forms both in 2D and 3D. NSCs cultured in 2D cultures on 4RepCT “film” structures showed no significant differences in cell proliferation, viability, or differentiation potentialcompared to control cultures in optimized conditions. 4RepCT substrates generated as “foam” structurescould be used for 3D culturing of NSCs, and these NSC cultures differentiated nicely into astrocytesand neurons. Calcium imaging assays revealed that BMP4+Wnt3a-treatment of NSCs grown in 3D4RepCT-matrices resulted in efficient generation of functional excitatory neurons. These studies have thus revealed new molecular mechanisms underlying neural differentiation ofcortical stem cells, and point to the versatility of using spider silk protein-based substrates for stem cellcultures. Future studies aim at testing these new conceptsin vivo for improved treatment of neurological disease

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+)-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture

The oxytocin/vasopressin receptor family has at least five members in the gnathostome lineage, inclucing two distinct V2 subtypes

The vertebrate oxytocin and vasopressin receptors form a family of G-protein-coupled receptors (GPCRs) that mediate a large variety of functions, including social behavior and the regulation of blood pressure, water balance and reproduction. In mammals four family members have been identified, three of which respond to vasopressin (VP) named V1A, V1B and V2, and one of which is activated by oxytocin (OT), called the OT receptor. Four receptors have been identified in chicken as well, but these have received different names. Until recently only V1-type receptors have been described in several species of teleost fishes. We have identified family members in several gnathostome genomes and performed phylogenetic analyses to classify OT/VP-receptors across species and determine orthology relationships. Our phylogenetic tree identifies five distinct ancestral gnathostome receptor subtypes in the OT/VP receptor family: V1A, V1B, V2A, V2B and OT receptors. The existence of distinct V2A and V2B receptors has not been previously recognized. We have found these two subtypes in all examined teleost genomes as well as in available frog and lizard genomes and conclude that the V2A-type is orthologous to mammalian V2 receptors whereas the V2B-type is orthologous to avian V2 receptors. Some teleost fishes have acquired additional and more recent gene duplicates with up to eight receptor family members. Thus, this analysis reveals an unprecedented complexity in the gnathostome repertoire of OT/VP receptors, opening interesting research avenues regarding functions such as regulation of water balance, reproduction and behavior, particularly in reptiles, amphibians, teleost fishes and cartilaginous fishes

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

The oxytocin/vasopressin receptor family has at least five members in the gnathostome lineage, including two distinct V2 subtypes

<p><strong>Research article: The oxytocin/vasopressin receptor family has at least five members in the gnathostome lineage, including two distinct V2 subtypes</strong></p> <p>Daniel Ocampo Daza*, Michalina Lewicka¹, Dan Larhammar<br>Department of Neuroscience, Science for Life Laboratory, Uppsala Universitet, Box 593, SE-751 24 Uppsala, Sweden</p> <p>* Corresponding author. E-mail address: [email protected]<br>¹ Current address: Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden</p> <p><em>General and Comparative Endocrinology 175(1): 135-143</em><br><em>doi:10.1016/j.ygcen.2011.10.011</em></p> <p>Accepted October 20, 2011<br>E-pub October 28, 2012<br>Published January 1, 2012</p> <p>This PDF and Supplementary material corresponds to the article as it appeared upon acceptance.</p> <p>Cite original work as <em>D. Ocampo Daza, M. Lewicka and D. Larhammar. The oxytocin/vasopressin family has at least five members in the gnathostome lineage, including two distinct V2 subtypes. General and Comparative Endocrinology, 175 (1) (2012) 135-143.</em></p> <p> </p