3 methyladenine MNs can be obtained from iPSCs
MNs can be obtained from iPSCs, using signaling molecules such as retinoic 3 methyladenine (RA) and Sonic hedgehog (Shh) (Table S1).4, 5, 12, 23, 24, 25, 26, 27, 28, 29 These methods rely on developmental principles and require changing the combinations of signaling molecules at multiple steps, which is why some methods require more than 4 weeks to produce MNs. In contrast, Hester et al. reported a rapid differentiation method using adenoviral vectors that encode the transcription factors neurogenin 2 (Ngn2), islet-1 (Isl1), and LIM/homeobox protein 3 (Lhx3). These three transcription factors were transduced into neural progenitor cells, and MNs were obtained 11 days after the transduction. Son et al. reported that mouse and human fibroblasts were converted directly into MNs using seven and eight transcription factors, respectively, encoded by retrovirus vectors. In 2013, Mazzoni et al. generated doxycyclin-inducible mouse embryonic stem cell lines to obtain MNs (Table S2). Methods that rely on transcription factors are simple and rapid; but, when we use them for research on MNDs, we have to consider the possibility of genomic integration of the vector genes. Vector gene integration into host genomes contains the risk of influencing the behaviors of the transduced cells. Moreover, when we transduce several transcription factors separately, the transduction ratio of each transcription factor varies between the cells, and the heterogeneity of the cells may influence the experimental results. Therefore, we decided to focus on Sendai virus (SeV) vectors33, 34 (Table S3), which never integrate into host genomes with highly efficient transduction and expression levels of the transgene(s), and we designed a single SeV vector that encodes Lhx3, Ngn2, and Isl1 to produce more homogeneous MNs. Here we report that MNs can be induced from ESCs/iPSCs using a single SeV vector encoding a combination of transcription factors and that ALS iPSC-derived MNs exhibit disease phenotypes.
Discussion Along with the development of stem cell technology, stem cell-derived MNs have been utilized for modeling MNDs in vitro. However, the heterogeneity of these MN populations presents a potential issue for disease modeling and analysis. To obtain more homogeneous MNs, we used a single SeV vector that encodes three transcription factors. SeV, known as murine parainfluenza virus type 1, is a negative sense, single-stranded RNA virus of the family Paramyxoviridae. SeV vectors are cytoplasmic RNA vectors that do not integrate into host genomes. They can be transduced into both dividing and non-dividing cells, and short-term exposure is enough for efficient transduction. SeV vectors can accommodate up to 5 kb of insertion.
Materials and Methods
Conflicts of Interest
Acknowledgments We would like to express our sincere gratitude to all our coworkers and collaborators, including Noriko Endo, Mayumi Yamada, and Ruri Taniguchi for their valuable administrative support, and Takumi Kanaya, Takeo Yamamoto, Kaoru Takizawa, and Takashi Hironaka for their valuable technical support. We acknowledge Peter Karagiannis for providing critical reading. Funding for this project was received in part from the Program for Intractable Diseases Research utilizing disease-specific iPSCs from Japan Agency for Medical Research and Development (AMED) to H.I., from the Research Project for Practical Applications of Regenerative Medicine from AMED to H.I., from the grant for Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine from AMED to H.I., and from the Daiichi Sankyo Foundation of Life Science to H.I.
Introduction Evolutionary conservation has become more and more a powerful tool to identify functionally important sequences in the genome (Dermitzakis et al., 2005). In this context, the ultraconserved elements (UCEs) are 481 genomic segments longer than 200 base pairs (bp), which are fully conserved (100% identity with no insertions or deletions) between human, mouse, and rat genomes (Bejerano et al., 2004). This complete conservation led to the hypothesis that UCEs likely have biological functions fundamental to mammal cells (Katzman et al., 2007). Despite extensive studies, our knowledge of UCEs is still limited. Indeed, increasing evidences indicate that UCEs play different functions in vertebrate genomes, acting as enhancer (Paparidis et al., 2007, Pennacchio et al., 2006), splicing (Ni et al., 2007), and epigenetic regulators (Bernstein et al., 2006, Lee et al., 2006), or functioning as transcriptional coactivators (Feng et al., 2006). In particular, many UCEs act as long-range enhancers during mouse development (Pennacchio et al., 2006), and it has been proposed that their removal in vivo would lead to a significant phenotypic impact. Nevertheless, knockout studies performed so far indicate that UCEs are dispensable for mice viability (Ahituv et al., 2007, Nobrega et al., 2004).