Isolation And Extraction Of Adipose Stem Cells And Induction Of Differentiation Into Schwann Cells

Objectives:The primary adipose-derived stem cells(ADSCs)of SD rats extracted by type I collagenase digestion were induced to differentiate into Schwann-like cells.The morphology,staining characteristics,cell surface characteristic molecules,purinergic receptor P2X7 and the neurotrophic factors and corresponding receptors were compared before and after differentiation of ADSCs.So as to observe whether ADSCs have the differentiatal potential to Schwann cells(SCs)and possess necessary materials for repairing nerve damage,provide experimental evidence for clinical ADSCs utility of nerve repairment.Methods:Male Sprague-Dawley rats weighing 300±10 g were dissected under sterile conditions to obtain adipose tissue in the inguinal region.After digestion with type I collagenase,the cells were centrifuged,filtered through a 200-mesh cell sieve,and inoculated.The cells were purified by differential adherence and passaged.The cell viability of the 3rd,5th,7th,9th and 13th generations were examined by the MTT assay.The third generation ADSCs cells were selected for the experiment.Flow cytometry was used to detect the ADSCs surface characteristic molecules CD29,CD44,CD45 and CD90,and the characteristics of ADSCs were identified according to their positive expression rate.uADSCs were plated at a density of 2×10~5/mL,treated with 1 mM?-mercaptoethanol for 24 h,35 ng/mL all-trans retinoic acid for 72h,5 ng/mL PDGF,10 ng/mL bFGF,14 uM Forskolin,200ng/mL heregulin for 1 week to induce differentiation,the morphological changes of cells were observed by oil red O staining before and after differentiation.Proteins were extracted from uADSCs and dADSCs,and quantified by BCA,purinogic receptor P2X7,neurotrophic factor NT-3,NRG-1,neurotrophic factor receptor TrkC,ErbB-4,and myelin protein P0 content changes were detected by Western blot.Results:The primary cultured ADSCs adhered faster.After 24 hours,pseudo-foot were extended from the cells body.After 48 hours,the cells were polymorphic with star-shaped,polygonal,and irregular.After passage,the cells were mostly fusiform.According to the MTT test,there was no significant difference in cell growth activity after resuscitation,and cell activity at the third generation was better than that of the fifth generation.Flow cytometry analysis of ADSCs showed negative expression of CD45 and positive expression of CD29,CD44 and CD90.Oil red O staining revealed red staining of uADSCs and no red staining of dADSCs,indicating that fat characteristics decreased after uADSCs differentiated into dADSC.Western Blot assay showed P2X7 and myelin protein P0 expression levels were no difference between uADSCs and dADSCs.While neurotrophic factors NT-3,NRG-1,and neurotrophin receptor TrkC,ErbB-4 were highly expressed in dADSCs than those of uADSCs indicating that after the differentiation of ADSCs,the synthesis ablity of nervous growth factors were enhanced which may related to the promotion of nerve growth..Conclusion:After primary cultured ADSCs were induced to differentiated Schwann-like cells,the lipogenic phenotype was decreased,the expression of neurotrophic factors and their corresponding receptors were increased but no changes in purinogic receptor P2X7 and myelin protein P0,demonstrating that the main mechanisms of differentiated Schwann-like cells for nerve repairmen was related to enhanced synthesis of nervous growth factors.