Covalent growth factor tethering to direct neural stem cell differentiation and self-organization

Abstract

Tethered growth factors offer exciting new possibilities for guiding stem cell behavior. However, many of the current methods present substantial drawbacks which can limit their application and confound results. In this work, we developed a new method for the site-specific covalent immobilization of azide-tagged growth factors and investigated its utility in a model system for guiding neural stem cell (NSC) behavior. An engineered interferon-γ (IFN-γ) fusion protein was tagged with an N-terminal azide group, and immobilized to two different dibenzocyclooctyne-functionalized biomimetic polysaccharides (chitosan and hyaluronan). We successfully immobilized azide-tagged IFN-γ under a wide variety of reaction conditions, both in solution and to bulk hydrogels. To understand the interplay between surface chemistry and protein immobilization, we cultured primary rat NSCs on both materials and showed pronounced biological effects. Expectedly, immobilized IFN-γ increased neuronal differentiation on both materials. Expression of other lineage markers varied depending on the material, suggesting that the interplay of surface chemistry and protein immobilization plays a large role in nuanced cell behavior. We also investigated the bioactivity of immobilized IFN-γ in a 3D environment in vivo and found that it sparked the robust formation of neural tube-like structures from encapsulated NSCs. These findings support a wide range of potential uses for this approach and provide further evidence that adult NSCs are capable of self-organization when exposed to the proper microenvironment.

Facile Quenching and Spatial Patterning of Cylooctynes via Strain-Promoted Alkyne–Azide Cycloaddition of Inorganic Azides

“…The resulting negative patterning may frequently be helpful in generating complex patterns, adding a complementary tool to the powerful photo-patterning toolkit 31–33 . Potential applications include the patterning of hydrogels using SPAAC reagents with azide-conjugated bioactive reagents such as growth or cell adhesion factors, for use in cell cultures 28–30 .…”

Biomaterials for Sequestration of Growth Factors and Modulation of Cell Behavior

“…Differentiation into other lineages varied between chitosan and HA, once again demonstrating the complex interplay between different stimuli on the control of stem cell fate. [71] Notwithstanding the latest advances of these systems, the strategy of covalently binding GFs to their supporting structure has raised some concerns. Conceptually, its chief aim is to avoid the "burst release" behavior often observed when GFs were physically adsorbed onto carrier systems, as well as to mimic the juxtacrine signaling mechanisms of the natural ECM.…”

Neurogenic Niche Conversion Strategy Induces Migration and Functional Neuronal Differentiation of Neural Precursor Cells Following Brain Injury

“…S4). Because the fate of NP/SCs depends on their microenvironment [42], the different fates of SVZ-derived NP/SCs between the control group and the treatment group may be attributable to different microenvironments within the injured cortex.…”