Concurrent Delivery of Soluble and Immobilized Proteins to Recruit and Differentiate Neural Stem Cells

Insufficient endogenous neural stem cell (NSC) migration to injury sites and incomplete replenishment of neurons complicates recovery following central nervous system (CNS) injury. Such insufficient migration can be addressed by delivering soluble chemotactic factors, such as stromal cell-derived factor 1-α (SDF-1α), to sites of injury. However, simply enhancing NSC migration is likely to result in insufficient regeneration, as the cells need to be given additional signals. Immobilized proteins, such as interferon-γ (IFN-γ) can encourage neurogenic differentiation of NSCs. Here, we combined both protein delivery paradigms: soluble SDF-1α delivery to enhance NSC migration alongside covalently tethered IFN-γ to differentiate the recruited NSCs into neurons. To slow the release of soluble SDF-1α, we copolymerized methacrylated heparin with methacrylamide chitosan (MAC), to which we tethered IFN-γ. We found that this hydrogel system could result in soft hydrogels with a ratio of up to 70:30 MAC/heparin by mass, which enabled the continuous release of SDF-1α over a period of 2 weeks. The hydrogels recruited NSCs in vitro over 2 weeks, proportional to their release rate: the 70:30 heparin gels recruited a consistent number of NSCs at each time point, while the formulations with less heparin recruited NSCs at only early time points. After remaining in contact with the hydrogels for 8 days, NSCs successfully differentiated into neurons. CNS regeneration is a complex challenge, and this system provides a foundation to address multiple aspects of that challenge.