Investigating Mechanisms of Subcutaneous Preconditioning Incubation for Neural Stem Cell Embedded Hydrogels

Stem cells are a vital component of regenerative medicine therapies, however, only a fraction of stem cells delivered to the central nervous system following injury survive the inflammatory environment. Previously, we showed that subcutaneous preconditioning of neural stem cell (NSC) embedded hydrogels for 28 days improved spinal cord injury (SCI) functional outcomes over controls. Here, we investigated the mechanism of subcutaneous preconditioning of NSC-embedded hydrogels, with and without the known neurogenic cue, interferon gamma (IFN gamma), for 3, 14, or 28 days to refine and identify subcutaneous preconditioning conditions by measurement of neurogenic markers and cytokines. Studying the preconditioning mechanism, we found that subcutaneous foreign body response (FBR) associated cytokines infiltrated the scaffold in groups with and without NSCs, with time point effects. A pro-inflammatory environment with upregulated interleukin (IL)-6, IL-10, macrophage inflammatory protein (MIP)-1, MIP-2, IFN-gamma-inducible protein 10 (IP-10), tumor necrosis factor-alpha (TNF-alpha), and IL-12p70 was observed on day 3. By 14 and 28 days, there was an increase in proregenerative cytokines (IL-13, IL-4) along with pro-inflammatory markers IL-1 beta, IP-10, and RANTES (regulated on activation, normal T cell expressed, and secreted) potentially part of the mechanism that had an increased functional outcome in SCI. Coinciding with changes in cytokines, the macrophage population increased over time from 3 to 28 days, whereas neutrophils peaked at 3 days with a significant decrease at later time points. Expression of the neuronal marker beta III tubulin in differentiating NSCs was supported at 3 days in the presence of soluble and immobilized IFN-gamma and at 14 days by immobilized IFN-gamma only, but it was greatly attenuated in all conditions at 28 days, partially because of dilution via host cell infiltration. We conclude that subcutaneously incubating NSC seeded scaffolds for 3 or 14 days could act as host specific preconditioning through exposure to FBR while retaining tubulin expression of NSCs to further improve the SCI functional outcome observed with 28 day subcutaneous incubation.