Differentiation Of Rat Bone Marrow-Derived Mesenchymal Stem Cells Into Dopaminergic Neurons In Vitro

Objective:TO investigate the isolation, culture and expansion of rat bone marrow-derived mesenchymal stem cells(MSCs)in vitro. To explore the condition of rat MSCs how to differentiate into dopamine (DA) neurons. we studied the effects of 2-mercaptoethanol (2-ME),ascorbic acid (AA) and glial cell line-derived neurotrophic factor (GDNF) on the differentiation of MSCs into DA neurons in vitro alone or combinely. To provide the experimental bases for the directional differentiation of dopaminergic (DA) neurons.Methods: MSCs were isolated from SD rat bone marrow by gradient centrifugation. The whole medulloculture plus digestion control for the separation, and serial subcultivation of MSCs.Morphology and growth characteristics were observed under inverted phase-contrast microscope. Ploting the growth curve to observe the capacity of self-renewing and proliferation of MSCs.And the MSCs were identified by inducing their differentiation to bone.P 3(passage 3) MSCs were plated on to the pre-differentiation medium supplemented with 1mmol/L2-ME + 10% Fetal bovine serum(FBS).After 24 hours, the cultured cells were washed for three times in the freezing phosphate -buffered saline (PBS) ,then they were induced in the differentiation and serum-free medium supplemented with of 5mmol/L2-ME + 100μmol/LAA + 10ng/mlGDNF①or 5mmol/l2-ME + 100μmol/LAA②or 5mmol/L2-ME + 10ng/mlGDNF③respectively for 5 hours. The specific antigens of DA neurons including tyrosine hydroxylase (TH) and dopamine transporter (DAT) and the neuron special enolase (NSE) antigen of normal neurons were detected by Immunocytochemistry, and TH mRNA was detected by RT-PCR.Results: (1) The highly purified SD rat bone marrow-derived MSCs of rats were harvested and the corpuscular activity was well keeping in this study.In vitro the bone marrow-derived MSCs were grewed with adherence and exhibited a spindle-shaped fibroblastic morphology. After bone induction, alizarin Bordeaux S showed that the calcium nodus was positive. (2)2-ME+100μmol/LAA, 2-ME+10ng/mlGDNF and 2-ME+ 100μmol/LAA+10ng/mlGDNF could make MSCs differentiate into TH-positive cells, DAT-positive cells and NSE-positive cells. In three group, NSE-positive cells, TH-positive cells and DAT -positive cells of the third group is the hightest. Each group could detect the expression of TH mRNA.Conclusion: (1) The culture method combining the whole medulloculture with digestion control is simply performed in need of simple condition,so it is one of the optimal methods for cell preparation and purification at present.The cultured MSCs are active in high purity with stable biological character in vitro. The MSCs have the capacity of self-renewing ,expansion and proliferation in vitro. The cultured cells could differentiate into bone in the condition.(2) The MSCs have the capacity of differenting into dopaminergic neurons. All the three groups could promote the differentiation of MSCs into DA neurons. When 2-ME,GDNF cooperated with AA, the effect was stronger than the other groups.