Covalent immobilization of azide-tagged interferon-gamma for the generation of adult stem cell-derived neuroepithelium

摘要

Introduction: Following CNS injury, cells within the damaged region experience rapid and sustained population loss. The post-injury activation of endogenous neural stem cells (NSCs) is insufficient; subsequently, adult CNS tissue is unable to regenerate. Delivering undifferentiated SCs is ineffective: the cells need a carrier material and the necessary cues to guide lineage commitment. Previously we have shown that interferon-γ (IFN-γ), immobilized to methacrylamide chitosan (MAC) hydrogels, provokes a potent neurogenic response when cultured with NSCs. In order to immobilize proteins to in situ hydrogels and more precisely direct NSC behavior, we engineered a new, bioorthogonal method of covalent immobilization (Fig. 1A and 1B). In this work, we present a new method for immobilizing N-terminally tagged IFN-γ via a Cu-free click reaction and explore applications for CNS repair. Methods: IFN-γ was produced recombinantly and tagged with an N-terminal azide using a double vector system. The tag was confirmed by MALDI and a fluorescent reporter assay. MAC was functionalized with dibenzocyclooctyne-NHS (DIBO-NHS) to yield MAC-DIBO and probed using FTIR. MAC-DIBO was reacted with azIFN-γ and then formed into hydrogels, which were washed and digested. The remaining azIFN-γ was measured to determine efficiency. Solutions of azIFN-γ or IFN-γ and varying amounts of BSA were incubated with hydrogels for 4d; efficiency was measured. The biological effects of i-azIFN-γ were measured by incubating NSCs on 2D gels in vitro for 8d. NSCs were also encapsulated within 3D gels and implanted subcutaneously for 4wks. In both cases, IHC and qPCR were performed to investigate NSC differentiation. AzIFN-γ was also immobilized to hyaluronan (HA) to show that its application is not limited to MAC. Results/Discussion: Both MALDI and the reporter assay indicated that the N-terminal tag was incorporated into azIFN-γ. FTIR showed a reduction in the 1° amine peak and concomitant appearance of an alkyne peak, demonstrating that MAC is functionalized at the 1° amines with DIBO as expected. An efficiency of 68.5 ± 2.74% was observed when reacting azIFN-γ with MAC-DIBO before hydrogel formation (dashed line in Fig. 1C). Excitingly, reacting azIFN-γ with pre-formed hydrogels showed similar efficiencies; the presence of competing protein (BSA) had no effect (Fig. 1B). This shows that azIFN-γ immobilization is bioorthogonal and that this system could be used to immobilize proteins to an implanted hydrogel at different timepoints in situ. A potent neurogenic response to i-azIFN-γ was observed in both in vitro culture and in vivo implantation (Fig. 2). Conclusion: azIFN-γ was covalently immobilized to MAC and HA. This system was shown to be bioorthogonal and capable of immobilizing IFN-γ to intact hydrogels with a powerful neurogenic response. This is especially important in vivo, as it allows us to precisely orchestrate important cellular responses at definable timepoints.