Establishing HiPSC-derived Neural Induction Model And Analysising Neurobiological Parameters Of Induced-neurons

Objective: To establish human pluripotent stem cells(hiPSC)derived neurons in vitro,and to the utilities of this in vitro model in mechanistic studies of neurodevelopment.Methods: 1)Neural development model was constructed by using dual inhibitor factors.Development of neuron morphology and function were monitored using immunofluorescence staining,electrophysiology,PCR and other methods throughout the neural induction course;2)immunofluorescence staining was used to identify the type of neurons,and Neurolucida software was used to construct and quantify neuronal morphology;3)Neuronal differentiation efficiency and the proportion of the neuronal subtypes were evaluated in different growth conditions(PDL + laminin,PDL + matrigel and matrigel coating),followed by quantification through unbiased ImageJ morphometric analyses.Results: 1)Our hiPSC cell-derived neurons can differentiate into GABAergic,dopaminergic,glutamatergic neurons,all of which exhibit morphological and functional characteristics of mature neurons;2)Transcriptome sequencing during developmental courses revealed that wnt2 B and wnt7 A in the WNT gene family were up-regulated in D0-D7 and down-regulated in D7-D28,whereas wnt5 A was down-regulated in D0-D7 and up-regulated in D7-D28;3)Our hiPSC cell differentiation and culture conditions favor the production of GABAergic neurons;the average proportion of glutamatergic neuron across cultures is 10.7%,dopaminergic neuron is about 17.3%,and GABAergic neuron is about 54.9%.The neurite lengths of GABAergic and glutamatergic neurons reached the peak at the third week in culture,and the development of dopaminergic neurons is throughout the entire process of neurodevelopment.The dendritic arborization of GABAergic neuron is primarily distributed within 100 ?m from soma,and glutamatergic and dopaminergic neurons are concentrated within 150 ?m.4)PDL+laminin coating conditions can improve the neuronal induction efficiency,comparing with PDL+matrigel and matrigel coating conditions,while coating conditions don't affect neuron subtype specification.Conclusions: 1)The neural induction model derived from human pluripotent stem cells was established successfully in this study;2)The related genes in the WNT gene family show differential expression during development time course,which suggests that WNT-related genes may be potential factors regulating neural differentiation and development;3)PDL+ Laminin coating condition can improve the efficiency of neuronal differentiation.