Immobilized ECM molecules and the effects of concentration and surface type on the control of NSC differentiation

Abstract

New developments in growth factor and pharmaceutical control of cells for neural regenerative strategies have built a need for high throughput assays to conveniently screen these treatments in vitro before moving to more complex experiments. Towards this application, we have studied an easy and highly reproducible culture regime with a renewable cell source for central nervous system (CNS) strategies. Adult neural stem cells (NSCs) derived from the CNS are attractive for use in screening assay because they are easily expanded and can differentiate into all major CNS cell types. NSCs were cultured on glass alone or methacrylamide chitosan (MAC) hydrogel coated glass substrates, immobilized with extracellular matrix (ECM) proteins collagen and laminin at varying densities. Proteins were also adsorbed on the surfaces as a control. We found that adsorbed protein on hydrogel coated glass resulted in the highest cell densities after 8 d, over twice the density of immobilized groups or adsorbed protein on glass. No significant differences were observed between collagen, laminin, or both proteins together regarding cell differentiation (p > 0.05); however, the morphological spreading and branching of differentiated NSC processes was enhanced on MAC substrates with covalently immobilized laminin protein. The results of this study suggest that a soft MAC hydrogel surface with adsorbed protein would be most desirable for specifying neuronal differentiation of large numbers of stem cells due to the high cellularities they supported.

Olfactory Ensheathing Cells Promote Differentiation of Neural Stem Cells and Robust Neurite Extension

“…Laminin is also critical for promoting neural progenitor differentiation 12 . Fibronectin with a molecular weight of 440 kDa has also been shown to promote neural progenitor migration 67–69 , although its role in neural differentiation is not documented in the manner than laminin has been.…”

“…OECs are a type of glial cell found in the lamina propria, a layer of tissue directly adjacent to the olfactory epithelium 8 and the olfactory bulb 9–12 . The olfactory epithelium is unique in the peripheral nervous system because it hosts the periodic neurogenesis of olfactory receptor neurons (ORNs) which are often damaged through the binding of odorants.…”

Engineering Biomaterials to Influence Oligodendroglial Growth, Maturation, and Myelin Production

“…While polymer hydrogels, such as those based on PEG, have been utilized ex-tensively for neural tissue engineering [Gunn et al, 2005;Burdick et al, 2006;Lampe et al, 2010b;Zustiak et al, 2013], no existing research identifies any oligodendroglial cell interactions. Working with methylcellulose and chitosan, the Shoichet and Leipzig groups have shown the importance of material stiffness on oligodendrocyte development [Leipzig and Shoichet, 2009;Wilkinson et al, 2014]. In our own lab with a purely synthetic methacrylated PEG hydrogel, we have similarly found that the material stiffness is important for OPC cell line proliferation [Liu et al, 2011] ( fig.…”

“…They also found that in conditions that had a larger concentration of βIII-tubulin, NG2-positive cells were at a higher concentration. Imaging also found that NG2-positive cells were extending processes on the hydrogel [Wilkinson et al, 2014].…”

“…More recent work has synthesized methacrylamide chitosan hydrogels to investigate cell differentiation on a 2D surface [Wilkinson et al, 2014]. Here, coverslips were first functionalized with methacrylates by allowing the self-assembly of 3-(trimethoxysilyl)propyl on the surface.…”