Differentiation Of Mesenchymal Stem Cells Derived From Rat Bone Marrow And Human Placenta Into Dopaminergic Cells

1.To culture and amplify mesenchymal stem cells from rat bone marrow and human placenta.2.To investigate the possibility of differerntiating mesenchymal stem cells into dopaminergic cells in vitro.Methods:1.Isolation,cultivation and identification of rBMMSCs and hPMSCs1.1.Mesenchymal stem cells were isolated by adhesive screening method from rat bone marrow and by digestion,adhesive screening method from human placenta,then cultured with DMEM/F12 medium supplemented with 10%fetal bovines serum(FBS).1.2.Expression of hPMSCs surface antigens was detected by flow cytometry.1.3.rBMMSCs和hPMSCs were cultured in adipogenic medium,The presence of adipocytes was assessed by the cellular accumulation of neutral lipid vacuoles that stained with Oil Red O. rBMMSCs和hPMSCs were cultured in an osteogenic medium,The onset of osteoblasts was evaluated by the expression of alkaline phosphatase.2.Differentiation and identification of rBMMSCs and hPMSCs into dopaminergic cell2.1.Differentiation of rBMMSCs into dopaminergic cellBasic fibroblast growth factor(bFGF),epidermal growth factor(EGF),antiscorbic acid(AA) were used for rBMMSCs to be induced into dopaminergic cells.2.2.Differentiation ofhPMSCs into dopaminergic cellProtocol 1(bFGF,EGF,AA),protocol2(bFGF,Ratinoic acid(RA),AA,3-isobutyl-1-methylx anthine(IBMX)) and protocol3(bFGF,β-mercaptoethanol(β-ME),dexame-thasone,insulin,nerve growth factor(NGF))were used for hPMSCs to be induced into dopaminergic cells.2.3.RT-PCR was used to determine the expression of gene nestin,nuclearrelate dreceptorl (nurr-1),aromatic L-amino acid decarboxylase(aadc),tyrosine hydroxylase(th) of rBMMSCs and the expression of gene nestin,tyrosine hydroxylase of hPMSCs.2.4.Immunofiuorescence staining was used to identify the expression of protein NESTIN, neuronal nuclear antigen(NeuN),dopamine transporter(DAT) and TH.2.5.The quantity of dopamine secretion and the content of intracellular dopamine were examined by enzyme-labeled immunosorbent assay(ELISA). Result:1.The correlated results of rBMMSCs(1)It took about 10-14 days for the primary rBMMSCs culture.Exposure of these cells to osteogenic inductive agents for 4w resulted in an osteoblast-like phenotype and the expression of alkaline phophatase.Moreover,when rBMMSCs were exposed to adipogenic medium for 2w,cells differentiated into adipocytes which displayed a perinuclear accumulation of lipid vacuoles.(2)Differentiation and identification of rBMMSCs into dopaminergic cell:genes related to nerves cell,such as nestin,nurr-1 were expressed when rBMMSCs were exposed to dopaminergic medium for 3d.when rBMMSCs were exposed to dopaminergic medium for 2w, genes aadc,th,nestin and nurrl were expressed,rBMMSCs before induction only expressedβ-actin.Immunofluorescence staining showed that NESTIN positive cells can be found on the third day of differentiation and TH positive cells can be found after seven days of differentiation,then DAT positive cells can be found after ten days of differentiation.ELISA demonstrated that Intracellular dopamine can be synthesized and secreted into supemate after dopaminergic differentiation.2.The correlated results of hPMSCs(1)It took about 21-25 days for the primary hPMSCs culture,knmunophenotyping of hPMSCs revealed these cells to be positive for CD44 and CD29,At the same time hPMSCs were negative for CD34,CD45,CD106 and HLA-DR.Exposure of these cells to osteogenic inductive agents for 4w resulted in an osteoblast-like phenotype and the expression of alkaline phophatase.When hPMSCs were exposed to an adipogenic medium for 4w,cells differentiated into adipacytes which displayed a perinuclear accumulation of lipid vacuoles.(2)Differentiation and identification of hPMSCs into dopaminergic cell:genes such as nestin and th were expressed after hPMSCs were exposed to dopaminergic medium and hPMSCs before induction were weak expressed nestin.Immunofluorescence staining showed that NEST1N positive cells can be found on the fifth day of differentiation,and TH positive cells can be found after seven days of differentiation,then DAT positive cells can be found after ten days of differentiation. ELISA demonstrated that Intracellular dopamine can be synthesized and secreted into supernate after dopaminergic differentiation.Conclusions: 1.MSCs from human placenta can be cultured and expanded in vitro.Immunophenotyping of hPMSCs revealed these cells to be positive for CD44 and CD29 and negative for CD34,CD45, CD106,HLA-DR.2.MSCs from rat bone marrow and human placenta have the ability to differentiate into adipocyte and osteoblast in vitro.3.MSCs from rat bone marrow and human placenta can be differentiated into dopaminergic cells in vitro,the differentiated dopaminergic cells can synthesize and secrete doparnine.