| Technical advances in the propagation and manipulation of mouse ES cells have improved our understanding of their growth and differentiation,but there are few reports about differentiated cholinergic neuronal cells and effects of their microenvironments and niches.In this study,the classical embryoid body(EB) 4-/4+ methods were carried out to obtain the mature neuronal cells derived from mouse ES cells and expressing varairation of Junctophilin 3,4(JP-3,4) during the process of differentiation.We would further investigate the effects of ICT on ES cells niches and their cholinergic phenotypes.The mitochondria-related molecular mechanisms were futher explored.Genes or proteins associated with development(OCT3/4),blastoderm(Fgf5,αFP, Branchy),neural phenotypes(nestin,β-tubulinⅢ) and The motor- and cholinergic function(CHAT,mAChR1-5) were detected to experience a variation as the cell maturation.Memory-related genes and proteins of JP-3,4 were verified expressed in the neurodifferentiative duration as well.However,the co-culture of hippocampus tissue and EBs could not enhance the neurogenesis of mouse ES cells.Neural progenitors generated from ICT-treated EBs were further differentiated into the neurons and also enhanced the choline acetyltransferase(CHAT) expression upon terminal differentiation,while the underlying mechanisms were not known yet. ICT possessed the ability to facilitate the mitochondria formation and sustain the normal△ψm by JC-1 staining and it may not cause cell apoptosis according to DAPI staining.Take together,RA possessed the neural inductive effect to enhance mouse ES cells differentiating to cholinergic neurons.The generated cells may express JP-3,4, but RA can not maintain the ratio of functioned cells.ICT could enhance cholinergic neuronal differentiation from mouse ES cells.It also possessed the ability to facilitate the mitochondria formation and sustain the normal△ψm.
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