Differentiated Human Adipose-Derived Stromal Cells Exhibit The Phenotypic Of Mature Schwann Cells And Promote Peripheral Nerve Regeneration Through A Modified Approach

Objective: Tissue engineering technology is a promising therapeutic strategy in peripheral nerve injury.Schwann cells(SCs)are deemed to be a vital component of cell-based nerve regeneration therapies.Many methods for producing SC-like cells derived from adipose-derived stromal cells(ADSCs)have been explored,but their phenotypic and functional characteristics remain unsatisfactory.Meanwhile,these characteristics cannot sustain for long periods of time.In SCs culture,insulin,progesterone(PROG)and glucocorticoids(GLUC)can promote myelination and neurotrophic synthesis.In the present study,we investigated whether original differentiation medium(ODM)combined with insulin,PROG and GLUC can induce ADSCs to differentiate into modified SC-like cells with better phenotypic and functional characteristics.Methods: In this research,we identified human ADSCs by flow cytometry analysis,osteogenic and adipogenic differentiation.Modified differentiation medium(MDM)was optimized by adding insulin,PROG and GLUC into ODM.The phenotypic and functional characteristics of original and modified SC-like cells were evaluated by real-time quantitative polymerase chain reaction(RT-PCR),enzyme-linked immunosorbent assay(Elisa)and immunofluorescence in vitro.The ability of proliferation and migration,SC phenotypic stability and the expression of neurotrophic factor of original and modified SC-like cells were assessed by MTT,Transwell assay,RT-PCR,Elisa and Western blot after withdrawing the induction reagents.Cells loaded into collagen sponge biomaterials were implanted around transected sciatic nerves with a 10-mm gap in vivo.Immunofluorescence,Gastrocnemius wet weight test,Masson’s trichrome staining assay and Walking footprint analysis were performed to evaluate axon regrowth and functional recovery of the regenerated nerves.Results: We have harvested and identified pure human ADSCs.After differentiation induction,the modified SC-like cells showed significantly up-regulated levels of S100 B and P0 and enhanced proliferative and migratory capacities.In addition,compared with original SC-like cells,the modified SC-like cells showed increased secretion of neurotrophic factors,and their functional characteristics were maintained for more than 3 weeks after removing the induction reagents.Tissue engineering nerves significantly promoted the regeneration of sciatic nerve in Sprague-Dawley rats after injury.The modified SC-like cells exhibited significantly enhanced axon regrowth,myelination.The potential mechanism may be the secretion of neurotrophic factors.and functional recovery after sciatic nerve injury.At 16 weeks post-operation,The sciatic nerves of rats implanted modified SC-like cells exhibited significantly greater functional improvement.The differences are statistically significant(P<0.05).Conclusion: Overall,the results suggest that this modified induction method can induce human ADSCs to differentiate into cells with the molecular and functional properties of mature SCs,and the properties can be maintained long after removal of the induction medium.Tissue-engineered nerves which we constructed can increased the promotion of peripheral nerve regeneration.This study provides a new experimental and theoretical basis for future clinical treatment.