Abstract
Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders. However, the molecular mechanisms underlying the neural commitment in humans remain elusive. Here, we report the establishment of a neural differentiation system based on human embryonic stem cells (hESCs) and on comprehensive RNA sequencing analysis of transcriptome dynamics during early hESC differentiation. Using weighted gene co-expression network analysis, we reveal that the hESC neurodevelopmental trajectory has five stages: pluripotency (day 0); differentiation initiation (days 2, 4, and 6); neural commitment (days 8-10); neural progenitor cell proliferation (days 12, 14, and 16); and neuronal differentiation (days 18, 20, and 22). These stages were characterized by unique module genes, which may recapitulate the early human cortical development. Moreover, a comparison of our RNA-sequencing data with several other transcriptome profiling datasets from mice and humans indicated that Module 3 associated with the day 8-10 stage is a critical window of fate switch from the pluripotency to the neural lineage. Interestingly, at this stage, no key extrinsic signals were activated. In contrast, using CRISPR/Cas9-mediated gene knockouts, we also found that intrinsic hub transcription factors, including the schizophrenia-associated SIX3 gene and septo-optic dysplasia-related HESX1 gene, are required to program hESC neural determination. Our results improve the understanding of the mechanism of neural commitment in the human brain and may help elucidate the etiology of human mental disorders and advance therapies for managing these conditions.
Highlights
Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders
By specific co-expression gene assays of transcriptome data with 12 samples prepared every other day between differentiation day 0 and day 22, we show that the following five distinct stages exist during the early neural differentiation of human embryonic stem cells (hESCs): pluripotency; differentiation initiation; neural commitment; NPC proliferation; and neuronal differentiation stage
Directed differentiation of hESCs mimics the early cortical development in vivo To investigate the regulatory mechanisms of human neural commitment, we first adapted the previous protocols [12] and standardized an in vitro hESC (H9 line) neural differentiation system, with EB formation for 6 days, attached EB for 10 days, sphere in N2 for 6 days, and single cells replated in N2B27 for 4 weeks (Fig. 1A)
Summary
Directed differentiation of hESCs mimics the early cortical development in vivo To investigate the regulatory mechanisms of human neural commitment, we first adapted the previous protocols [12] and standardized an in vitro hESC (H9 line) neural differentiation system, with EB formation for 6 days, attached EB (aEB) for 10 days, sphere in N2 for 6 days, and single cells replated in N2B27 for 4 weeks (Fig. 1A). Clustering analysis using the highest PC-loading genes in the PC1 [24] (top 100 PC1-negative and -positive genes) suggested that day 8 in cluster 3 is a critical fate transition period during hESC neural differentiation (supplemental Fig. S2E). The dynamic gene expression profiles revealed a temporal developmental trajectory of hESC neural differentiation, and day 8/10 of cluster 3 is a critical fate transition period. RT-qPCR assays revealed that at differentiation day 12, the expression of pluripotent genes OCT4 and NANOG was increased, but the neural epithelium marker genes OTX2, PAX6, SOX1, and ZIC1 was significantly reduced (Fig. 6B). Similar results were obtained in another clone of SIX3 and HESX1 deletion cells (supplemental Fig. S7) Taken together, these data suggest that the neural differentiation of hESCs is compromised in the lossof-function of either the SIX3 or HESX1 gene. These results suggest that SIX3 and HESX1 genes may intrinsically promote neural differentiation by regulating its downstream TF networks
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