Abstract
The generation and use of induced pluripotent stem cells (iPSCs) in order to obtain all differentiated adult cell morphologies without requiring embryonic stem cells is one of the most important discoveries in molecular biology. Among the uses of iPSCs is the generation of neuron cells and organoids to study the biological cues underlying neuronal and brain development, in addition to neurological diseases. These iPSC-derived neuronal differentiation models allow us to examine the gene regulatory factors involved in such processes. Among these regulatory factors are long non-coding RNAs (lncRNAs), genes that are transcribed from the genome and have key biological functions in establishing phenotypes, but are frequently not included in studies focusing on protein coding genes. Here, we provide a comprehensive analysis and overview of the coding and non-coding transcriptome during multiple stages of the iPSC-derived neuronal differentiation process using RNA-seq. We identify previously unannotated lncRNAs via genome-guided de novo transcriptome assembly, and the distinct characteristics of the transcriptome during each stage, including differentially expressed and stage specific genes. We further identify key genes of the human neuronal differentiation network, representing novel candidates likely to have critical roles in neurogenesis using coexpression network analysis. Our findings provide a valuable resource for future studies on neuronal differentiation.
Highlights
Our study proposes potential functions of annotated and novel long non-coding RNAs (lncRNAs) based on coexpression network and hub gene analysis, and provides a useful resource for further studies that examine the roles of lncRNAs in biological processes, such as mammalian development and neurogenesis
Human induced pluripotent stem cells (iPSCs) cultured in feeder-free monolayer conditions were exposed to the neural induction medium, and subsequently replated in neuronal progenitor medium (NPM)
Our findings revealed that the iPSC samples show the highest amount of differentially expressed genes (DEG), coding and non-coding, both in percentage of gene category (Figure 3A) and number of genes (Figure 3B), and throughout the differentiation late stages having a lower amount of DEGs between each other
Summary
Increasing number of studies have highlighted that pluripotent stem cell (ESC/iPSC) technologies provide a notable platform to generate specific types of neuron from healthy and patient-derived iPSCs, in vitro models to elucidate the biological cues of neuronal development and the cellular/. An integrative approach combining molecular biology and bioengineering approaches with computational biology methods has been implemented to overcome these limitations and generate reliable, functional in vitro models These in vitro models allow to investigate the transcriptome dynamics and characteristic parameters of generated cells during neuronal specification. Analysis of gene expression dynamics in human iPSC-derived neurons provide a solid framework to study early neural developmental process, progenitor differentiation, distinct axonal development (Compagnucci et al, 2015; Grassi et al, 2020; Lindhout et al, 2020). Our study proposes potential functions of annotated and novel lncRNAs based on coexpression network and hub gene analysis, and provides a useful resource for further studies that examine the roles of lncRNAs in biological processes, such as mammalian development and neurogenesis
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