Cell-free Fat Extract Promote Axon Regeneration: a Pre-clinical Study.

Abstract Background: Injured axons of the central nervous system (CNS) hardly regenerate due to their poor intrinsic regeneration ability and adverse microenvironment at the site of injury; previous studies aimed to investigate both the above-mentioned factors and achieved some success. Compared with the gene-editing technology, stem cell therapy has unique advantages, such as low risk of tumor formation and ease-of-use, among others. However, traditional stem cell therapy is hindered by safety and immunogenicity issues. Previously, we developed a cell-free extract directly from human fat tissue (CEFFE) and demonstrated its proangiogenic capacity. Herein, we aimed to evaluate its potential therapeutic effect in injured CNS.Methods: Therapeutic potential of CEFFE in injured CNS was investigated, using a mouse optic nerve crush model. After the optic nerve was crushed, CEFFE was injected intravitreally. Two weeks post-injury, the number of regenerated axons was measured at different distances from the crush site (250, 500, 750, 1000, and 1500 µm). In addition, surviving retinal ganglion cells (RGCs) were counted. Cellular locations and catalogs of CEFFE were analyzed by gene ontology (GO) annotation. Western blotting (WB) was performed to evaluate the therapeutic effects of CEFFE on the molecular level. An enzyme-linked immunosorbent assay (ELISA) was used to examine the levels of inflammatory factors in CEFFE. Results: Compared with brain-derived neurotrophic factor (BDNF), CEFFE effectively promoted axon regeneration and RGCs survival. GO annotation showed that 146 proteins were involved in axon genesis or neurogenesis, most of which were located intracellularly. WB and ELISA results showed that CEFFE acted on diverse pathways and involved high levels of inflammatory factors. Conclusions: The present findings have shown that CEFFE may promote axon regeneration and increase RGCs survival. The levels of factors involved in CEFFE were much higher than those observed in the traditional secretome; CEFFE involves multiple factors and affects several mechanisms. This study has revealeded the potential application of CEFFE for the treatment of CNS injury.