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 the developing forebrain however is still incomplete. Studies of embryonic telencephalic neural stem cells (NSCs) in vitro may 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 thesis is aimed at investigating the mechanisms underlying bone morphogenetic protein(BMP4)-mediated differentiation of NSCs, and to explore the use of recombinant spider silk protein-based matrices in combination with signaling factors, especially BMP4, to generate functional neural cell circuits in vitro. In the first study we discovered that BMP4 treatment of NSCs resulted in a dramatic increase in the expression of the BMP4-inhibitor Noggin. BMP4 mediated non-neural differentiation into mesenchymal cells at low seeding densities, neuronal differentiation at high seeding densities, and astrocyte differentiation in any condition. As the Noggin levels increased linearly at higher densities, we hypothesized that the endogenous Noggin production predominantly mediated an inhibition of mesenchymal differentiation. We further observed that BMP4 stimulation induced an AMPA responsive 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 GABAergic neurons, 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 a novel 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, and possibility 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 potential compared to control cultures in optimized conditions. 4RepCT substrates generated as “foam” structures could be used for 3D culturing of NSCs, and these NSC cultures differentiated nicely into astrocytes and 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 of cortical stem cells, and point to the versatility of using spider silk protein-based substrates for stem cell cultures. Future studies aim at testing these new concepts in vivo for improved treatment of neurological disease.

List of scientific papers

I. Andersson T, Duckworth JK, Fritz N, Lewicka M, Södersten E, Uhlén P, Hermanson O. (2011) Noggin and Wnt3a enable BMP-4 dependent differentiation of telencephalic stem cells into GluR-agonist responsive neurons. Mol Cell Neurosci. 47:10-8.
https://doi.org/10.1016/j.mcn.2011.01.006

II. Lewicka M, Rebellato P, Uhlén P, Hermanson O. (2013) BMP4-induced neuronal differentiation of cortical stem cells involves a Hes6-dependent GABAergic component. [Manuscript]

III. Lewicka M, Hermanson O, Rising AU. (2012) Recombinant spider silk matrices for neural stem cell cultures. Biomaterials. 33:7712-7717.
https://doi.org/10.1016/j.biomaterials.2012.07.021

IV. Lewicka M, Rebellato P, Uhlén P, Rising A, Hermanson O. (2013) Building brains in spider webs: Stem cell-derived GluR-agonist responsive neurons grown in 3D matrices of recombinant spider silk protein. [Manuscript]