Differentiation Of Human Bone Marrow Mesenchymal Stem Cells Into Cholinergic Neuron-like Cells In Vitro

Objective To explore the isolation,purification,amplification and differentiation towards cholinergic neuron-like cells of human bone marrow mesenchymal stem cells(BMMSCs) in vitro.Methods The BMMSCs were isolated from human bone marrow through density gradient centrifugation method, then amplified and cultivated in a total medium of DMEM/F12 containing 10%FBS,Surface antigen of BMMSCs were detected by flow cytometry. And the BMMSCs were divided into two groups in the second passage. In the experiment group, the BMMSCs were pretreated with a total mediem consisting of 40mg/L WHI-P131 for 48h and preinduced with 10ng/ml basic fibroblast growth factor(bFGF) for 24h,then induced with 1μmol/L alltransretinoic acid(ATRA) and 10ng/ml sonic hedgehog(Shh) for 5h.On the other hand,none of inductive agents were added in the control group.After the induction, The BMMSCs were observed using phase-contrast microscopy and detected by neuron specific enolase(NSE),glia fibrillary acid protein(GFAP) and choline acetyltransferase(ChAT) immunocytoch- emistry,then the percentages of NSE,GFAP and ChAT positive cells were calculated respectively.Results BMMSCs expressed strongly CD29,weakly CD44,CD166,CD105,did not express CD45,CD34,HLA-DR.Under induction conditions, most of the BMMSCs resumed typical neuronal morphology, and the percentages of NSE,GFAP and ChAT positive cells were(61.00±3.63)%(,14.42±2.74)% and (9.92±1.29)% respectively. But in the control group,the BMMSCs remained unchange morphologically and none of the them expressed NSE,GFAP and ChAT.Conclusion Human BMMSCs can be induced to differentiate towards neuron-like and cholinergic neuron-like cells in vitro.