Preliminary Study Of In Vitro Induced Differentiation Of Rat ADSCs Into Photoceptors And RPE Cells.

Part One:Isolation,culture and identification of rat ADSCsObject:To isolate,cultivate and identify rat ADSCsMethods:The ADSCs were isolated by TypeⅠCollagenase and cultured by their adherent ability to the wall of culture flask in DMEM/F12 medium supplemented with 10%fetal bovine serum.The ADSCs were purified by the method of continual passage.Surface antigens including CD45,CD90,CD49d,CD106 were indentified by flow cytometry.Results:Large amout of ADSCs were obtained with high purity.For primary generation,the phenotypes of ADSCs were CD45(1.6%),CD90(71.3%), CD49d(7.8%) and CD106(3.5%).From passage 1 to 5,these phenotypes were CD45(0.8%～9.3%),CD90(84.7%～94.8%),CD49d(16.8%～31.0%) and CD106(8.3%～22.2%).There was a higher CD49d percentage than CD106 in all the passages.Part Two:In vitro differentiation of rat ADSCs into photoreceptor cells and RPE cellsObject:To explore and compare the induced differentiation of rat ADSCs into Photoreceptor cells and RPE cells.Methods:Retinal neuroepithelial cells were isolated and obtained by mechanic measures and furthure digested by Dispase for RPE cells as well as extracted liquid of retina.ADSCs were induced for differentiation by EGF,activin A, Taurine,retinoic acid(RA) and extracted liquid of retina respectively. Meanwhile,ADSCs were combinedly induced by EGF+Taurine,EGF+RA, Taurine + RA,EGF + Taurine + RA respectively.Immunofluorescence was introduced for detecting the expression of rhodopsin,CK and S-100 and flow cytometry for quantification.Induction efficacy was calculated by contrasting control groups and induced groups.MSCs underwent the same procedures and served as reference.Results:A portion of the induced cells in the extracted liquid of retina were oval or fusiform,with pigment inside plasm.The induced cells in the 1%FBS/αMEM with induction agents presented a slower growth and flat polygonal shape.The induction efficacy of ADSCs was 17.5%～46.0%for rhodopsin,19.7%～79.3%for CK and 27.3%～50.7%for S-100.The induction efficacy of MSCs was 55.9%～76.5%for rhodopsin,39.4%～75.9%for CK and 25.4%～41.8% for S-100.Conclusion:More ADSCs will be otabined by modified isolation and culture of the primary generation.The characteristic adherence to the wall of culture flask facilitates high purity.Mechanic measures in the isolation of retinal neuroepithelial cells and Dispase digestion for RPE cells successfully prepares extracted liquid of retina which induced differentiation of ADSCs.After either single induction or combined induction,the ADSCs expressed the specific markers of photoceptors(rhodopsin) and RPE markers(CK and S-100),which indicated potential differentiation into photoceptors and RPE cells.ADSCs present a similar induction efficacy to MSCs.The novel method of extracted liquid of retina proves to be economical and effective in induced differentiation of ADSCs and MSCs.