Enhanced Derivation Of Human Pluripotent Stem Cell-derived Cortical Glutamatergic Neurons By A Small Molecular

Objective:This study application of SHH inhibitor cyclopamine to reduced GABAergic neurons and to promote the differentiation of glutamatergic neurons from hPSCs which may provide an efficient system for disease modeling and cell-based therapeutic treatment.Method:(1)we differentiated human embryonic stem cells(H9)to neural neuroepithelial cells according to our previously described method under a highly chemically controlled system.The ventral GABAergic neurons were detected by Immunostaining.(2)To determine the most effective concentrations of cyclopamine(Cyclo),we respenctively applied 0 μM,2.5 μM or 5 pM to hESC-derived neural epithelium cultures from day 10 to day 25.The efficiency concentrations of cyclopamine were compared by qPCR,Immunostaining,western blotting and Glutamate Assay Kit(3)To investigate the optimal time window of inhibiting LGE progenitors,we added 5 μM cyclopamine starting at day 1,day 7 or day 10,with continuous treatment to day 25.The efficiency time window of cyclopamine were compared by qPCR and Immunostaining.(4)To gain insight into DS,we applied our current optimized protocol to differentiate cortical glutamatergic neurons from the trisomy iPSC line(DS1)and its isogenic control iPSCs line(DS2U).The differences were compared by Immunostaining Results:(1)Indeed,numbers of GABAergic neurons related markers were determined after day 35 and GABA neurons co-expressed the LGE marker MEIS2,suggesting ventral origin.These results were consistent with those of previous studies reporting the differentiation of telencephalic progenitors in the absence of exogenous morphogens yielding glutamatergic neurons with a mixture of numbers of LGE GABAergic neurons.(2)5μM cyclopamine was the optimal condition for the inhibition of ventral cells.(3)Early treatment of cyclopamine promoted the generation of glutamatergic neurons and decreased the GABAergic neurons.(4)With the application of 5 pM cyclopamine starting at day 1,trisomy iPSC(DS1)-derived neurons exhibited decreased BNPI+puncta compared to the disomy(DS2U)control.However,the two groups did not show significant differences in the absence of cyclopamine.Conclusion:(1)Application of 5 μM cyclopamine from day 1 to day 25 during differentiation efficiently promoted dorsal patterning.(2)Cyclopamine promoted the generation of glutamatergic neurons,and may aid glutamatergic neuron-based disease studies using DS iPSCs.