Embryonic stem cell (ESC) in vitro neural differentiation has been widely utilized to study the regulation of neurogenesis. On the other hand, it also provides feasible access to neural stem cells (NSC) or neurons in the cell replacement therapy of neural degenerative diseases. Different protocols have been developed to derive and propagate neural stem cells from ESCs cultured in vitro. However, most of these NSCs are lack of true neural stem cell properties，i.e. they displayed differentiation bias and failed to generate all cell types of neural lineage, or they lost the ability to respond to regional cue .Elkabetz et al. identified an earlier stage of NSC population during the process of human ESC neural differentiation. R-NSC possesses true NSC properties and is considered as stem cells of neural lineage. Albeit potential in the study of neurogenesis as well as in cell replacement therapy, R-NSC is difficult to be maintained and propagated in culture. Moreover, whether R-NSC exhibits high risk of tumorigenesis following transplantation remains elusive. To overcome these obstacles, intensive study of R-NSC is required in order to understand the molecular mechanisms regulating the self-renewal and multipotency of these cells. However, few work was undertaken and little information was obtained regarding the molecular properties of R-NSC in human and even in mice. Rhesus monkey (Macaca mulatta) has more than 90% (92.5% to 95%) DNA homolog to human and has long been considered as a reliable non-human primate model to study various human diseases and to assess the preclinical safety of medical treatments. In the study, we examined by deep sequencing the dynamic transcriptome and miRNAome changes during the differentiation of rhesus embryonic stem cells (rESCs) into the neural lineages. In the study, we examined by deep sequencing the dynamic transcriptome and miRNAome changes during the differentiation of rhesus embryonic stem cells (rESCs) into the neural lineages—neural rosette cells passage 1 (R-NSCP1), R-NSC passage 6 (R-NSCP6), and neural progenitor cells (NPCs). We identified Hedgehog signaling pathway are closely associated with the transition from ESCs to R-NSCP1. Besides, many alternative splicing switches were also found during state transitions. Referring to miRNAomes, many miRNAs were found to be absent in NPCs, leading to the activities of some signaling pathways, such as Wnt signaling pathway. Besides, we also detected miRNAs with high expression variations among lineages, which could be used to maintain specific stages during the neural differentiation.Through the comprehensive bioinformatic analysis of these data, we identified a set of genes as stage-specific markers of neural differentiation, most of which were conserved in human ESC neural differentiation. Many alternative splicing switches were observed during the variable stage transition. We also identified a list of miRNAs with drastic expression level change along with the neural differentiation. The data provided valuable and comprehensive information to further understand the molecular basis regulating the neural differentiation of primate pluripotent stem cells. The candidate genes and microRNAs specifically or highly expressed in R-NSC identified in this study ultimately providing therapeutic solutions for neural degenerative diseases.Keywords: Rhesus monkey; embryonic stem cells; Neural differentiation; Neural stem cells, RNA-seq; Transcriptome; miRNAomes
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