LXR Agonist Promotes Differentiation Of Rat Bone Marrow Derived Mesenchymal Stem Cells Into Dopaminergic Neurons

Objective: To observe the effect of liver X receptors(LXR)agonist on the differentiation of rat bone marrow derived mesenchymal stem cells(BMSCs)into dopaminergic(DA)neurons and its possible mechanism.Methods: 1.The method of whole bone marrow adherence was used to isolate,purify and culture of BMSCs from SD rats.To identify the BMSCs,osteogenesis and adipogenesis were used to identify multiple differentiation potential of BMSCs,and the expressions of CD45,CD11 b,CD44,CD90 and CD34 were measured by flow cytometry.2.BMSCs from rat were divided into 4 groups,including control group,LXR group,Growth factors treated group(GF group)and LXR agonist and growth factors treated group(LXR+GF group).(1)Determine for the adding time of LXR agonist: Based on the method of GF group,0.5 ?M LXR agonist was added into the medium at 3-day intervals for 9 days.The expressions of Tuj1 and TH were measured by immunocytochemistry.(2)Determine for the time for induction period of LXR agonist: 0.5 ?M LXR agonist and GF were added into the medium according to the result of(1)and cells' statuses were observed at 3-day intervals.The expressions of Tuj1 and TH were measured by immunocytochemistry.(3)Determine for the effect-concentration relationship of LXR agonist: Different concentrations of LXR agonist(0.125,0.25,0.5,1 and 2 ?M)were added with GF into the medium according to the results of(2)and(3).The expressions of Nestin,Neun,Tuj1 and TH were measured by immunocytochemistry.The growth rate was measured by CCK-8 assay.3.BMSCs from rat were divided into 3 groups,including control group,GF group and GF+LXR group(the simultaneous addition of GF+LXR(0.5 ?M)treated 6 days).The protein and mRNA expressions of LXR ? and LXR ? were measured by immunocytochemistry and qPCR,respectively.The mRNA expressions of ABCA1,TH,DAT,Nurr1,Pitx3,En1 and Lmx1 b were measured by q PCR.Results: 1.BMSCs that isolated,purified and cultured from SD rat were spindle cell-based with abundant cytoplasm,large nucleia and prominent nucleoli,growing in a radial colony arrangement way.BMSCs could be induced differentiattion into adipogenesis and osteogenesis.BMSCs negatively expressed CD45 and CD11 b and positively expressed CD44,CD29 and CD90.2.Cells in control group and LXR group were typical BMSCs morphology i.e.very flat,symmetrical,and spindle-shaped.The growth rate in both control group and LXR group was increased gradually and without significant difference between the two groups.Almost cells in control group have no expression of Nestin,Neun,TH and Tuj1.The results indicated that cells treated with LXR agonist alone had no effect on cell proliferation and differentiation.Expressions of Tuj1 and TH reached the peak at simultaneous addition of LXR agonist and growth factors(Day0).Accompanying with the adding time of LXR agonist delayed,expressions of Tuj1 and TH were decreased gradually.Simultaneous addition of LXR agonist and growth factors showed expressions of Tuj1 and TH reached the peak at day 6 and the growth rate of cells in GF group and LXR+GF group reached the peak at 6 days.Expressions of Tuj1 and TH increased significantly compared with those of GF group.Cells in GF group and simultaneous addition of LXR agonist and growth factors group revealed the appearance of extended long cellular processes and retracted cell bodies,which were the typical neuronal morphology.Cells in different concentrations of LXR agonist showed the typical neuronal morphology.Maximal expression of TH was obtained at concentration of 0.5 ?M.Compared with control group,a total number of TH positive cells were 62.61±2.334%.Compared with GF group,a total number of TH positive cells were 87.42±7.573% in LXR+GF group.3.Compared with control group,treatment of GF significantly increased the expressions of LXR ?,ABCA1,TH,DAT,Nurr1,and Pitx3 mRNA and significantly decreased the expressions of En1 and Lmx1 b mRNA.Compared with GF group,simultaneous addition of LXR and GF for 6 days,the protein and mRNA expressions of LXR ? and LXR ? and the mRNA expressions of ABCA1,TH,DAT,Nurr1,and Pitx3 significantly increased,and the expressions of Lmx1 b and En1 mRNA significantly decreased.Conclusions: 1.LXR agonist alone did not induce the differentiation of rat BMSCs into DA neurons.2.LXR activation leads to improve induction efficiency and shorten induction period of rat BMSCs into DA neurons.The optimal induced protocol was that simultaneous addition of LXR agonist and GF induced rat BMSCs to TH-expressing cells in 6 days at the most appropriate concentration of 0.5 ?M.3.The mechanism of LXR agonist promoting differentiation of rat BMSCs into DA neurons may be involved in regulating its downstream ABCA1 and DA development-related genes expressions by LXR activation.