Effects Of Different Feeder Cells On Dopaminergic Differentiation Of Human Embryonic Stem Cells

Background and Objection:Mouse embryonic fibroblasts(MEFs)and human foreskin fibroblasts(HFFs)are widely used for the culture of human embryonic stem cells(hESCs).MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs.The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines.MethodsTo minimize the impact of culture condition variation,two hESCs lines were cultured on mixed feeder cells(MFCs,MEFs:HFFs D 1:1)and HFFs feeder,respectively.The process of differentiation was performed under the condition of feeder-free culture system.The induction of dopaminergic differentiation was initiated by dual inhibition of SMAD pathway.The differentiated neurons were analyzed by phase contrast microscope,immunocytochemistry,real-time fluorescent quantitative PCR,transmission electron microscopy,scanning electron microscopy and patch clamp.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:The whole morphology of HN4 and P96 hESCs had changed from cobblestone-like shape to typical neuron shape.The differentiated cells of HN4 and P96 cell lines expressed the dopaminergic marker TH.The proportion of TH positive cells in P96-derived cells was higher than that of HN4-derived cells(P<0.05).The external morphological features,internal ultrastructures and synaptic connection of HN4-,P96-derived DA neurons were obtained by SEM and TEM.The P96-derived cell population had significantly higher expression levels of FOXA2,TH,PITX3 and NURR1 in comparison to HN4-derived cell population,particularly FOXA2 level,which showed significant(P<0.05)difference between the two groups.The presence of functional voltage-gated Na+ and K+ channels,GABA-and Glu-evoked currents,and spontaneous postsynaptic currents was observed in HN4 and P96 hESCs-derived neurons in the voltage-clamp mode.Both HN4-derived neurons and P96-derived neurons were able to fire trains of action potentials in current-clamp mode.A linear relationship between the number of spikes elicited and the intensity of the depolarizing stimulus was found in P96 or HN4-derived neurons.The duration of the AP was>2 mS,respectively.Resting membrane potential of HN4-and P96-derived DA neurons is-63.38 ± 0.01 mV and-58.7 ± 0.04 mV.Spike threshold of APs was-39.58 ± 0.17 mV for P96-derived DA neurons,whereas it was-36.15 ± 0.34 mV for HN4-derived DA neurons.These differences showed significant(P<0.05).The human ESCs-derived neurons demonstrated the ability of repetitive firing at later stage of differentiation in response to depolarizing current injection.The similar repetitive burst firing patterns in response to current injection were observed in P96 or HN4-derived neurons.The AP train evoked by depolarizing current revealed that each successive spike was a progressive increase in AP duration and spike threshold,a decline in AP amplitude,termed burst firing pattern.Conclusion1.The two hESCs lines derived on MEFs and HFFs feeder cells,HN4 and P96,were differentiated into morphology chemically fully maturated DA neurons with extensive dendrites and multiple synaptic contacts,and formed complex networks.The efficiency of DA neuronal differentiation of HN4 cell line was higher than that of P96 cell line(P<0.05).2.The two hESCs lines derived on MEFs and HFFs feeder cells,HN4 and P96,were differentiated into electrophysiological matured DA neurons.P96-derived DA neurons exhibited higher excitability.