Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation

Abstract

Background Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Results Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Conclusions Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

Identification of Therapeutic Targets for Amyotrophic Lateral Sclerosis Using PandaOmics – An AI-Enabled Biological Target Discovery Platform

“…While future studies are warranted to assess the pathogenic mechanisms underlying every stage of disease development, data generated in the current analysis is likely representing two stages in ALS development - the late-stage in post-mortem CNS tissue, and the early-stage reflected in diMN samples, harvested on Day 32. This time frame is generally adopted as the maturation time point of diMNs ( Du et al, 2015 ; Guo et al, 2017 ; Solomon et al, 2021 ); therefore, this study model might not well represent the aging effect on ALS disease progression. Including additional diMN time points throughout the developmental stages of the disease might enhance the comprehensiveness of the analysis and provide a more detailed insight on ALS progression.…”

Transcriptome Dynamics of Human Neuronal Differentiation From iPSC

“…We also inspected independent iPSC-derived neuronal differentiation datasets to observe whether reads in the datasets aligned to the same transcripts. To do so, we utilized two studies on iPSC-derived neuron transcriptomes that used a comparable sequencing depth and sequencing platforms, as well as ribosomal RNA depleted sequencing libraries ( Burke et al, 2020 ; Solomon et al, 2021 ). We found that out of the 354 identified lncRNA candidates, 350 showed expression in at least one sample of the independent datasets, and 296 showed expression in one sample in both datasets ( Supplementary Files S2, S3 ).…”

“…Purple cells indicate increased expression compared to the mean, while green cells indicate decreased expression. Dataset originally described by Solomon et al (2021) .…”

The Relationship Between Psychological Quality Education and Mental Health Level of College Students by Educational Psychology

“…Therefore, educators should view the psychological problems of college students from a development perspective, analyze the problems they face through multiple angles, and make positive interpretations. Secondly, development and correction are relative ( Eicher et al, 2018 ; Solomon et al, 2021 ). From the perspective of educational psychology, the focus of college students’ psychological quality education is to develop the potential ability of college students, and put the prevention and treatment of mental diseases in an auxiliary position.…”