Abstract
The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-β and canonical WNT/β-CATENIN signaling, confirming the roles of classical signaling in human neural induction. Interestingly, WNT/Ca2+ signaling was upregulated. Pharmacological inhibition of the downstream effector of WNT/Ca2+ pathway, Ca2+/calmodulin-dependent protein kinase II (CaMKII), led to an inhibition of the neural marker PAX6 and upregulation of epidermal marker K18, suggesting that Ca2+/CaMKII signaling promotes neural induction by preventing the alternative epidermal fate. In addition, our analyses revealed known and novel expression patterns of genes that are involved in DNA methylation, histone modification, as well as epithelial-mesenchymal transition, highlighting potential roles of those genes and signaling pathways during neural differentiation.
Highlights
Embryonic stem cells (ESCs), isolated from the inner cell mass of a preimplantation embryo, can differentiate to all cell types of the body, including neural cells[8]
Our time-course microarray along hESC neural differentiation enables us to interrogate the dynamics of global gene expression and signaling pathways that are involved in early neural differentiation
Gene ontology analysis reveals potential roles of less well-studied pathways in neural induction, including Epithelial-mesenchymal transition (EMT), as indicated by specific expression patterns of extracellular matrix protein and adhesion genes during the transition period, and epigenetic regulation, as indicated by a characteristic expression pattern of genes that are involved in DNA methylation and histone modification
Summary
Embryonic stem cells (ESCs), isolated from the inner cell mass of a preimplantation embryo, can differentiate to all cell types of the body, including neural cells[8]. Uniform ESC population is converted to highly enriched (~90%) neuroepithelia in 10 days, which enables gene expression profiles to reflect the respective cell types. Day 6 is a critical stage by which hESCs transition to early neuroepithelia Molecular profiling of these three stages would reveal dynamic changes in gene expressions and signaling pathways that are associated with the cellular conversion from ESCs to NE. Our study revealed known and novel expression patterns of genes that are involved in forebrain development, DNA methylation, histone modification, as well as epithelial-mesenchymal transition, which are validated with qRT-PCR. These findings highlight potential unique roles of those genes and signaling pathways during neural induction. Pathway analyses revealed upregulation of WNT/Ca2+ signaling and pharmacological inhibition of the downstream effector of WNT/Ca2+ pathway, Ca2+/calmodulin-dependent protein kinase II (CaMKII) resulted in inhibition of the neural but upregulation of epidermal markers, suggesting a role of non-canonical WNT signaling in partitioning the neural vs. epidermal fate during neural induction
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