Efficient Generation Of Neural Precursors From Rat Adipose-derived Stem Cells

Objective: The derivation of highly enriched populations of neural precursors fromrat ADSCs. Rat adipose derived stem cells (ADSCs)were transfected by lentiviralvector containing Green Fluorescent Protein (GFP), To observe the transfectionefficiency of lentiviral vector and to detect Lineage differentiation to adipogenic cellphenotype and osteogenic cell phenotype from transfected ADSCs.Methods: Rat ADSCs were cultured in serum-free neurobasal medium with theaddition of20ng/ml epidermal growth factor,20ng/ml fibroblast growth factor-2,2%B27，either on uncoated culture flasks or on coated culture flasks. Transfectionefficiency was assessed by GFP expression under fluorescence microscope afterADSCs were transfected48hours. Alizarin red staining and oil red O staining wereused to detect adipogenic and osteogenic differentiation ofADSCs after transfection.Results: While on uncoated culture flasks, rat ADSCs can be differentiated intoneural precursors efficiently. These cells grow in neurosphere-like structures, expresshigh levels of neural stem cells markers (Nestin). However, on coated culture flasks,only a small number of NPs were formed. In the abscence of growth factors,ADSCs-derived NPs can be differentiated into neuronal and glial phenotypes, expressneuron and astroglia markers (NeuN, GFAP). Upon differentiation, the expression ofNestin becomes downregulated. Transfection efficiency was85%after rat ADSCswere transfected by lentiviral vector containing Green Fluorescent Protein(GFP);They do express CD29(99.8%),CD45(48.7%),but the expression ofCD34(0.3%)and CD44(0.3%) is low; Adipocytes containing lipid droplets detected byoil Red O staining; The mineralized nodule became reddish-brown by alizarin red staining.Conclusion: Our results demonstrate that the presence of ADSCs-derived NPs arehighly enriched in absence of any attachment factor and were cultured in serum-freeneurobasal medium with the addition of20ng/ml epidermal growth factor,20ng/mlfibroblast growth factor-2,2%B27. ADSCs-derived NPs have potential forautologous cell transplantation therapy for neurological disorders in the future.ADSCs transfected by lentiviral vectors carrying GFP can effectively infect ADSCs,viral infection could not affect multi-directional differentiation of ADSCs, whichsuggested that lentiviral vectors carrying targeting gene can be used for gene therapy.