Three-Dimensional Motor Nerve Organoid Generation

Abstract

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

eIF2B-capturing viral protein NSs suppresses the integrated stress response

“…Considering that ISRIB shows beneficial effects in various neuropathological models (Halliday et al, 2015; Chou et al, 2017; Zhu et al, 2019; Bugallo et al, 2020), it is tempting to speculate that SFSV NSs also effectively works in such conditions. To assess the neuroprotective activity of SFSV NSs under the ISR-inducible stress conditions, we prepared primary cultures of hippocampal neurons from rat embryos, and motor neurons differentiated from human induced pluripotent stem (iPS) cells (Osaki et al, 2020) (Figure 5). The Tg treatment of rat primary hippocampal neurons decreased the length of their neurites, as expected (Figures 5A and 5B).…”

“…The reduced availability of the eIF2•GTP•Met-tRNA i ternary complex evokes "leaky" scanning to skip the uORFs and then drives translation from the downstream main ORF. ATF4, a stress-inducible transcription factor, is a remarkable example of translational stimulation through uORFs (Pakos-Zebrucka et al, 2016). Indeed, upon Tg treatment, the translation from the main ATF4 ORF was increased (Figures 4D, 4E, and S4A).…”

“…To further investigate the net alteration of translation over the transcriptome, which may be the ultimate consequence of ER stress (Pakos-Zebrucka et al, 2016), we evaluated the translational efficiency by the ribosome footprint abundance normalized by RNA sequencing (RNA-Seq). The translation efficiency changes in the ISR were not uniform among the transcripts, and a group of mRNAs, such as ribosomal protein mRNAs, were highly sensitive to Tg (Figures 4D and S3D).…”

“…The cells were maintained in Neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B27 supplement (Gibco), 1% GlutaMAX, and 1% penicillin/streptomycin). After 2 weeks, thapsigargin (Tg, 500 nM) was added to the culture medium for either 3 or 24 h. The motor neuron differentiation protocol has been described previously (Osaki et al, 2020). Briefly, human induced pluripotent stem cells (409B2 from the RIKEN Cell Bank) were seeded in Matrigel-coated plates at 70–80% confluency, in mTeSR Plus medium (STEMCELL Technology) with 10 μM of Y-23632 (Rock inhibitor, Wako).…”

“…In vertebrates, four protein kinases, PKR, PERK, GCN2, and HRI, which are activated by different stress stimuli, commonly phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2). Diverse stress signals are thus integrated into the phosphorylation of eIF2, and activate the common downstream pathway (Pakos-Zebrucka, et al, 2016). eIF2 is a heterotrimeric G-protein that delivers an initiator methionyl-tRNA (Met-tRNAi) to the 40S ribosomes in a GTP-dependent manner, and is also involved in the start codon recognition process on the ribosomes.…”