Angiogenic potential of co-spheroids of neural stem cells and endothelial cells in injectable gelatin-based hydrogel.

Appropriate crosstalk between neural stem cells (NSCs) and endothelial cells (ECs) is essential for establishment of the neurovascular network and neuroregeneration in the central nervous system (CNS) in vivo. However, platforms used to study the interaction of NSCs and ECs in three-dimensional (3D) environment are still rare. Here, we employed the chitosan-based substrates to rapidly generate the 3D NSC/EC co-spheroids in vitro, and then analyzed their crosstalk in the co-spheroids. By the analysis of gene and protein expression, NSCs in the NSC/EC co-spheroids displayed greater differentiation potential than the regular 2D co-culture on plastic dish. We also encapsulated the NSC/EC co-spheroids into chitosan- or gelatin-based hydrogels to further support the long-term growth of cell spheroids in a 3D environment. We observed that NSC/EC co-spheroids exhibited greater viability in the gelatin-based hydrogel, and even formed tube-like structures from the surface of the co-spheroids after FGF2 induction, indicating the increased angiogenic potential of ECs in the NSC/EC co-spheroids embedded in the FGF2-containing gelatin-based hydrogel. Finally, we demonstrated the injectability and printability of NSC/EC co-spheroids encapsulated in the gelatin-based hydrogel, revealing the possibility of using NSC/EC co-spheroids to build the biomimetic neurovascular constructs in the future.