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.
Highlights
Amyotrophic lateral sclerosis (ALS), the most common and severe form of motor neuron diseases (MNDs), causes progressive muscle weakness and leads to death within several years
On day 0, induced pluripotent stem cells (iPSCs) were seeded on Matrigel-coated dishes and the medium was changed from Embryonic stem cells (ESCs) medium to neurobasal medium with N2 and B27 supplements
To analyze the efficiency for the differentiation to motor neurons (MNs) from neural lineage cells, we transduced Sendai virus (SeV)-L-N-I-EGFP after treatment with dorsomorphin and SB431542 for 4 or 7 days (Figure 5). These results showed that the differentiation to MNs from neural lineage cells increased the number of HB9-positive cells compared to that from iPSCs
Summary
Amyotrophic lateral sclerosis (ALS), the most common and severe form of motor neuron diseases (MNDs), causes progressive muscle weakness and leads to death within several years. The only FDA-approved drug, riluzole, was reported to prolong patient lifespan by just a few months.[1] The establishment of induced pluripotent stem cells (iPSCs) offers a new approach to the study of MNDs and the discovery of new drugs.[2,3] In 2008, the first ALS patient iPSC-derived motor neurons (MNs) were generated.[4] Since many ALS iPSC studies have been reported,[4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22] and this technology is leading to new findings and therapeutic candidates for ALS
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