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

Abstract

Short title: Neuronal differentiation of neural stem cells in 3D spider silk matrices Abbreviations: NSCs (neural stem cells), BMP4 (Bone Morphogenic protein 4), TuJ1 (Neuronal class III B-tubulin), P (poly-ornithine), F (fibronectin), ECM (extracellular matrix), 3D (three dimensional), BW (BMP4+Wnt3a) 2 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 animalor 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.

Spider Silk for Tissue Engineering Applications

“…Results revealed that cells cultured on spider silk surfaces were capable of differentiating into neuronal and astrocytic Molecules 2020, 25, 737 15 of 20 cells without any significant differences to cells cultured on polystyrene plates with fibronectin coatings. Lewicka et al [89] also showed that the 3D porous foam structure made of 4RepCT supported NSC attachment, survival and differentiation into astrocytes in 3D. In this study, they cultured NSCs isolated from the cerebral cortices of rat embryos on 3D foams and monitored the interaction of the cells within the porous structure in terms of generation of functional excitatory neurons from NSCs without the addition of animal or human-derived components.…”

“…Application of stem cells and neuronal progenitors in contact with recombinant spider silk was studied by Lewicka et al [89]. In general, this type of cells is capable of differentiating into neurons, astrocytes, and oligodendrocytes.…”

“…The functionality of the differentiated neurons was tested after stimulating them with glutamate receptor agonists, such as α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor, and cells cultured on spider silk foams responded to at least the same extent as control cultures. Application of stem cells and neuronal progenitors in contact with recombinant spider silk was studied by Lewicka et al [89]. In general, this type of cells is capable of differentiating into neurons, astrocytes, and oligodendrocytes.…”

Host Systems for the Production of Recombinant Spider Silk

“…Notably, a recent study by Lewicka and co-workers reported that structures consisting of recombinant spidroins provide excellent matrices for the generation and analysis of stem cells and neuronal progenitors [71]. Moreover, DeSimone and colleagues have reported successful development of a hydrogel 'bioink' consisting of recombinant silk proteins encapsulating a…”

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

“…This work was funded by grants from CIMED, StratRegen (SFO to KI), Karolinska Institutets Forskningsstiftelser (2011FoBi023) (to AR), VR-MH (Project Grants to PU, AR, and OH), Karolinska Institutet (TEMA), the Swedish Childhood Cancer Foundation (BCF), the Swedish Cancer Society, and Vinnova (to OH). A previous version of this manuscript has been released as a pre-print at bioRxiv (Lewicka et al, 2019).…”