Simple Derivation of Spinal Motor Neurons from ESCs/iPSCs Using Sendai Virus Vectors

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal degenerative disorder of motor neurons (MNs). Embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) now help us to understand the pathomechanisms of ALS via disease modeling. Various methods to differentiate ESCs/iPSCs into MNs by the addition of signaling molecules have been reported. However, classical methods require multiple steps, and newer simple methods using the transduction of transcription factors run the risk of genomic integration of the vector genes. Heterogeneity of the expression levels of the transcription factors also remains an issue. Here we describe a novel approach for differentiating human and mouse ESCs/iPSCs into MNs using a single Sendai virus vector encoding three transcription factors, LIM/homeobox protein 3, neurogenin 2, and islet-1, which are integration free. This single-vector method, generating HB9-positive cells on day 2 from human iPSCs, increases the ratio of MNs to neurons compared to the use of three separate Sendai virus vectors. In addition, the MNs derived via this method from iPSCs of ALS patients and model mice display disease phenotypes. This simple approach significantly reduces the efforts required to generate MNs, and it provides a useful tool for disease modeling.

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

“…These results warrant future studies to elucidate the structural basis of the Atoh1/NM‐II interaction, and, based on this knowledge, to optimize miDA neuron production. Our unpublished results also support the interaction between Ngn2 and the NM‐II complex, suggesting that the NM‐II complex may interact with various proneural TFs, and NM‐II agonists may promote iPSC differentiation driven by various proneural TFs, such as Ngn2‐driven motor neuron differentiation .…”

Transcription Factor-Based Fate Specification and Forward Programming for Neural Regeneration

“…Yet, both MN subpopulations become electrophysiologically functional and capable of forming cholinergic synapses after maturation on cortical mouse astrocytes (Mazzoni et al, 2013). A few years later, Goto et al (2017) verified that Sendai virus-mediated overexpression of the TF cocktail NGN2, ISL1 and LHX3 in human PSCs, too, promotes the expression of MN markers. More specifically, only the full TF cocktail and the 2-factor combination of NGN2 and LHX3 but neither NGN2 in conjunction with ISL1 nor any of the single TFs resulted in MN derivation.…”

“…More specifically, only the full TF cocktail and the 2-factor combination of NGN2 and LHX3 but neither NGN2 in conjunction with ISL1 nor any of the single TFs resulted in MN derivation. Notably, after 3 weeks of differentiation NGN2/ISL1/LHX3overexpressing neurons were electrophysiologically active and formed neuromuscular junctions with cultured myocytes (Goto et al, 2017). De expressed both TF combinations identified by Hynek Wichterle's group (NGN2/ISL1/LHX3 and NGN2/ISL1/PHOX2A) in human iPSCs via Piggy-bac transposable vectors.…”

Transcription Factor–Mediated Differentiation of Human iPSCs into Neurons

“…Whereas small molecules elicit inconsistent effects based on cell‐to‐cell variability in uptake and metabolism, stably integrated transcription factors enable uniformly efficient differentiation. These strategies are gaining momentum in the iPSC field, with recent reports detailing TF‐based strategies for iPSC‐derived motor neurons, oligodendrocytes, and even pancreatic beta cells (Goto et al., ; Major, Powers, & Tabar, ; Zhu, Liu, Zhou, & Ikeda, ). Ultimately, TF‐based differentiation offers higher efficiency, higher purity, and a shorter timeline for producing the desired cell model when compared to small molecule differentiation methods.…”