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Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs.

Yoshida M, Kitaoka S, Egawa N, Yamane M, Ikeda R, Tsukita K, Amano N, Watanabe A, Morimoto M, Takahashi J, Hosoi H, Nakahata T, Inoue H, Saito MK - Stem Cell Reports (2015)

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.

View Article: PubMed Central - PubMed

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.

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The Pre- and Post-synaptic Morphological Defects in Type 1 SMA(A–D) Representative confocal micrographs showing the immunocytochemically labeled NMJ-LSs of iPSC-derived neurons and C2C12 myotubes. (A and B) Representative images of AChR clusters formed by C1 MNs. (C) AChR clusters stained with MHC. (D) NMJ-LSs of patient (P1)- and control (C1)-derived MNs on day 60.(E) Quantitative immunocytochemical analysis of the α-BTX-positive area (means ± SEM, n = 3, Wilcoxon rank-sum test).(F) Abnormal NF accumulation (yellow arrows) and poor terminal arborization of MNs in the SMA NMJ-LSs. See also Figure S2.
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fig2: The Pre- and Post-synaptic Morphological Defects in Type 1 SMA(A–D) Representative confocal micrographs showing the immunocytochemically labeled NMJ-LSs of iPSC-derived neurons and C2C12 myotubes. (A and B) Representative images of AChR clusters formed by C1 MNs. (C) AChR clusters stained with MHC. (D) NMJ-LSs of patient (P1)- and control (C1)-derived MNs on day 60.(E) Quantitative immunocytochemical analysis of the α-BTX-positive area (means ± SEM, n = 3, Wilcoxon rank-sum test).(F) Abnormal NF accumulation (yellow arrows) and poor terminal arborization of MNs in the SMA NMJ-LSs. See also Figure S2.

Mentions: We next tried to develop an in vitro NMJ formation model using the human iPSC-derived MNs. We co-cultured control MNs with differentiated murine C2C12 cell lines and found that αBTX-positive AChRs were clustered at the site of SV2-positive neuronal endplates (Figure 2A). To exclude the presence of unexpected artifacts of αBTX staining under these conditions, we co-stained samples with αBTX and anti-AChR antibodies and confirmed that the regions of both positive staining merged completely (Figure 2B). In addition, the AChR clusters were localized on myosin heavy chain (MHC)-positive multinuclear myotubes (Figure 2C).


Modeling the early phenotype at the neuromuscular junction of spinal muscular atrophy using patient-derived iPSCs.

Yoshida M, Kitaoka S, Egawa N, Yamane M, Ikeda R, Tsukita K, Amano N, Watanabe A, Morimoto M, Takahashi J, Hosoi H, Nakahata T, Inoue H, Saito MK - Stem Cell Reports (2015)

The Pre- and Post-synaptic Morphological Defects in Type 1 SMA(A–D) Representative confocal micrographs showing the immunocytochemically labeled NMJ-LSs of iPSC-derived neurons and C2C12 myotubes. (A and B) Representative images of AChR clusters formed by C1 MNs. (C) AChR clusters stained with MHC. (D) NMJ-LSs of patient (P1)- and control (C1)-derived MNs on day 60.(E) Quantitative immunocytochemical analysis of the α-BTX-positive area (means ± SEM, n = 3, Wilcoxon rank-sum test).(F) Abnormal NF accumulation (yellow arrows) and poor terminal arborization of MNs in the SMA NMJ-LSs. See also Figure S2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400613&req=5

fig2: The Pre- and Post-synaptic Morphological Defects in Type 1 SMA(A–D) Representative confocal micrographs showing the immunocytochemically labeled NMJ-LSs of iPSC-derived neurons and C2C12 myotubes. (A and B) Representative images of AChR clusters formed by C1 MNs. (C) AChR clusters stained with MHC. (D) NMJ-LSs of patient (P1)- and control (C1)-derived MNs on day 60.(E) Quantitative immunocytochemical analysis of the α-BTX-positive area (means ± SEM, n = 3, Wilcoxon rank-sum test).(F) Abnormal NF accumulation (yellow arrows) and poor terminal arborization of MNs in the SMA NMJ-LSs. See also Figure S2.
Mentions: We next tried to develop an in vitro NMJ formation model using the human iPSC-derived MNs. We co-cultured control MNs with differentiated murine C2C12 cell lines and found that αBTX-positive AChRs were clustered at the site of SV2-positive neuronal endplates (Figure 2A). To exclude the presence of unexpected artifacts of αBTX staining under these conditions, we co-stained samples with αBTX and anti-AChR antibodies and confirmed that the regions of both positive staining merged completely (Figure 2B). In addition, the AChR clusters were localized on myosin heavy chain (MHC)-positive multinuclear myotubes (Figure 2C).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus