Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs.
Bottom Line: We generated NMJ-like structures using MNs derived from SMA patient-specific induced pluripotent stem cells (iPSCs), and found that the clustering of the acetylcholine receptor (AChR) is significantly impaired.Valproic acid and antisense oligonucleotide treatment ameliorated the AChR clustering defects, leading to an increase in the level of full-length SMN transcripts.Thus, the current in vitro model of AChR clustering using SMA patient-derived iPSCs is useful to dissect the pathophysiological mechanisms underlying the development of SMA, and to evaluate the efficacy of new therapeutic approaches.
Affiliation: Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.Show MeSH
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Mentions: Fibroblasts from two independent type 1 SMA patients (Coriell IDs GM00232 and GM03813, referred to as P1 and P2) were reprogrammed by episomal vectors (Okita et al., 2011). Both SMA-iPSC clones used in this study (P1 and P2) showed a characteristic human embryonic stem cell (ESC)-like morphology, and expressed pluripotent markers compared to control ESC (KhES1) and iPSCs (201B7 and 409B2, referred to as C1 and C2) (Figures S1A and S1B). The RNA microarray analysis confirmed that the global gene expression pattern (Figures S1C and S1D) and levels of pluripotent stem cell-related genes (Figure S1E) in the P1 and P2 iPSCs were similar to that observed in the control iPSCs. The P1 and P2 iPSCs also exhibited demethylation of NANOG and OCT3/4 loci (Figure S1F) and maintained a normal karyotype (Figure S1G). Pluripotency of P1 and P2 iPSC lines were confirmed by teratoma formation assay (Figure S1H). The expression of introduced transgenes was rarely detected (Figure S1I). The genetic identity of the iPSC clones was proven by a short tandem repeat analysis (data not shown). SMA-iPSCs were confirmed to carry homozygosis deletions of exons 7 and 8 of the SMN1 gene (Figure 1A; van der Steege et al., 1995), and their SMN protein level was also significantly lower than that in control iPSCs, including C1 and C2 (Figures 1B and 1C).
Affiliation: Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Department of Pediatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.