Abstract
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.
Highlights
The microenvironment strongly influences stem cell characteristics in various ways
A recombinant spider silk protein (4RepCT; consisting of four tandem repeats and the C-terminal domain of the major ampullate spidroin 1) that corresponds to approximately 10% of the native spider silk protein, can be readily produced in Escherichia coli (E. coli) and spontaneously assembles into films, foams and fibers (Stark et al, 2007; Widhe et al, 2010). 4RepCT matrices provide excellent substrates for human primary fibroblasts (Widhe et al, 2010) and we have demonstrated that this recombinant spider silk protein provides a suitable substrate for culturing of rodent cortical neural stem cells (NSCs) as these cells proliferate, survive, retain multipotency, and differentiate into multiple cell types efficiently on film structures derived from 4RepCT without any additional coating or animal-derived components (Lewicka et al, 2012)
NSCs derived from rat cortex of embryonic day 15 (E15) were cultured in optimal conditions seeded on polystyrene cell culture plates precoated with poly-L-ornithine and fibronectin as described in Materials and Methods
Summary
The microenvironment strongly influences stem cell characteristics in various ways These effects include changes in differentiationassociated gene expression via epigenetic mechanisms, e.g., chromatin modifications, and related changes in transcriptional activity. As it has been shown repeatedly that the expression of differentiation markers not necessarily correlate with functionality (Andersson et al, 2011) such functional analysis is required before proceeding with transplantations and in vivo studies in animals
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