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    • Since we focused on the pathology of NMJ we

    2018-10-31

    Since we focused on the pathology of NMJ, we performed a detailed examination of our NMJ-LS. To evaluate whether the developmental status of the MNs affects the NMJ pathology, we evaluated AChR clustering at three different time points and obtained consistent data. In addition to impaired AChR clustering, we detected abnormal presynaptic NF accumulation at the endplate in the SMA-iPSC-derived neurons, which also indicates that synaptic breakdown precedes motor neuronal death in our model. These data support a csf1r that MNs derived from patient iPSCs are a major contributing factor to the pathogenesis in the NMJ due to SMA. Based on these observations, we considered that the morphological defect of NMJ-LS in our culture was due to functional impairments of the MNs in target pathfinding and/or in inducing or maintaining AChR clustering, rather than due to motor neuronal loss. Considering that the formation and maintenance of NMJs has been indicated to precede the occurrence of MN death even in humans, as mentioned above, the vulnerability of MNs in SMA patients seems to be due not only to the autonomous cell susceptibility to various stresses, but also as a consequence of the NMJ defect, which causes the impairment of neurotrophic factors and subsequent death of MNs (Fidziańska and Rafalowska, 2002; Fischer et al., 2004).
    Experimental Procedures
    Author Contributions
    Acknowledgments We are grateful to Y. Sasaki, Y. Jindai, S. Nakamura, S. Benno, and T. Ohkame for their technical assistance. We also thank A. Niwa, K. Oshima, T. Tanaka, and K. Chiyonobu for scientific comments, and H. Watanabe for administrative assistance. We are grateful to Dr. Keisuke Okita for plasmid distribution and scientific comments. This work was supported by grants from Ministry of Health, Labour and Welfare of Japan; grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Leading Project of Ministry of Education, Culture, Sports, Science and Technology (T.N.); the Funding Program for World-Leading Innovative Research and Development on Science and Technology of the Japan Society for the Promotion of Science (T.N. and M.K.S.); the grant for Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine from the Japan Science and Technology Agency (JST) (T.N., H.I, and M.K.S.); CREST (H.I.); the Ministry of Health, Labour and Welfare of Japan (H.I.); the Ministry of Education, Culture, Sports, Science and Technology of Japan (Innovative Area Foundation of Synapse and Neurocircuit Pathology [22110007] to H.I.); the Program for Intractable Diseases Research utilizing disease-specific iPS cells of JST (H.I. and T.N.); the Japan Research Foundation for Clinical Pharmacology (H.I.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research (H.I.); and Intramural Research Grant (24-9) for Neurological and Psychiatry Disorders of NCNP (H.I.).
    Introduction We focused our initial development of this therapeutic approach on cystic fibrosis (CF). The primary defect in CF, an autosomal recessive disorder, is the regulation of epithelial chloride transport by a chloride channel protein encoded by the CF transmembrane conductance regulator (CFTR) gene (Kerem et al., 1989). Recurrent pulmonary infections are responsible for 80%–90% of the deaths in CF patients. Therefore, transplantation of CFTR-corrected, autologous lung stem/progenitor cells provides an attractive alternative strategy for treating CF. Here we used zinc-finger nuclease (ZFN)-mediated HDR to edit the endogenous CFTR locus and precisely correct mutations responsible for CF in patient-derived iPSCs.
    Results
    Discussion We utilized ZFN-mediated gene editing (Urnov et al., 2005; Hockemeyer et al., 2009) to correct, in a site-specific manner, the CFTR mutation in iPSCs derived from CF patients. The generation of iPSCs from CF patients has been reported previously, with subsequent differentiation into epithelial cells (Somers et al., 2010; Mou et al., 2012; Wong et al., 2012; Sargent et al., 2014). The design and assessment of CFTR-specific nucleases also has been reported previously (Maeder et al., 2008; Lee et al., 2012; Sargent et al., 2014), including repair of the mutant CFTR gene. Site-specifically editing the endogenous gene (Garate et al., 2013) offers the potential for physiologically regulated expression of the therapeutic gene, retaining the expression of alternately spliced isoforms and eliminating the potential interfering influence of inherited mutations that may be dominant negative. In vitro differentiation of the mutant CF iPSCs into lung epithelial cells and tissue, controlled for by the parallel differentiation of the otherwise isogenic corrected CF iPSCs, may provide a valuable tool for examining the functional consequence of mutant CFTR expression. Furthermore, corrected CF iPSCs present a potential source of patient-specific cells capable, in vitro, of differentiation into various lung stem/progenitor cells (Weiss et al., 2011), either for transplantation of autologous lung cells or for seeding de-vitalized lung scaffolds ex vivo to generate autologous lungs (Ott et al., 2010).