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

Sammanfattning: 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.