Function Differentiation Of Dopaminergic Neurons Derived From Human Embryonic Stem Cells

Objection:On the basis of our differentiation protocol,we observated if dopaminergic neurons coming from human embryonic stem cell were mature in the function of morphology and electrophysiology.We assessed transplanted reliability of dopaminergic neurons,refering to the electrophysiological characteristics of substantia nigra compacta dopaminergic neurons in vivo.Methods:This research induced human embryonic stem cells H9 directionally differentiating to dopaminergic neurons,adopting the single-layer adherent method and the dual inhibition of BMP/SMAD channels.The differentiated neurons were analyzed by phase contrast microscope,immunocytochemistry,real-time fluorescent quantitative PCR,transmission electron microscopy,scanning electron microscopy and patch clamp.With reference to previous studies’ electrophysiology evaluation criteria of dopaminergic neurons in vivo,we evaluated the electrophysiological properties of H9 dopaminergic neurons.All of the statistical tests were performed using the Statistical Package SPSS version 19.0.Significance levels were set to P<0.05 for all comparisons.Results:In the course of differentiation in vitro,H9 showed the process from typical stem cells from to the typical neurons form: In the stage of embryonic stem cells,cells looked like paving stones with clear cytoplasm and high nucleocytoplasmic ratio;Intercellular space became tight gradually,and cells became smaller and rounder after switching the neural induction medium;Cells gathered into the shape of garland 16 th,then we picked clone suspending to neural ball which were uniform sizes and smooth texture;Nerve ball differentiated into typical neuron shape when switching the medium of promoting neurons mature 40 th.We choose Nestin to tab the neural stem cells which observed abundant.We choose the neurons marker MAP2,dopaminergic neurons marker TH and A9 dopaminergic neurons marker GIRK2 as specific markers.We observed the MAP2 / TH double positive cells and the TH/GIRK2 double positive cells,being dopaminergic neurons and A9 dopaminergic neurons with the differentiation efficiency of 28% and 18% respectively.We found products owning the typical morphology and ultrastructure of dopaminergic neurons including:organelles,cell nucleus,ribosomes,nerve and nerve synapses by the scanning electron microscopy and transmission electron microscopy technology.These results suggested that the products being differentiated from H9 were mature dopaminergic neurons.At the same time,we test the electrophysiology of neurons being differentiated from H9,and confirmed that the neurons had mature voltage-gated ion channels and ligand gating ion channels as same as the capacities to fire action potentials.We adopted the patch clamp electrophysiological technique to evaluate the electrophysiological function of dopaminergic neurons differentiated in vitro comparing with the electrophysiology standard of dopaminergic neurons in vivo.Conclusions:To sum up,this research adopted the BMP/SMAD dual channels inhibition to inducte H9 to differentiate into dopaminergic neurons which owned mature morphology and electrophysiological function.The dopaminergic neurons being differentiated from H9 conformed the electrophysiological characteristics of dopaminergic neurons in the substantia nigra compacta in vivo,which was good for the recovery of neural function and neural network after transplantation.