Recapitulating muscle disease phenotypes with myotonic dystrophy 1 induced pluripotent stem cells: a tool for disease modeling and drug discovery

Abstract

Myotonic dystrophy 1 (DM1) is a multisystem disorder primarily affecting the central nervous system, heart and skeletal muscle. It is caused by an expansion of the CTG trinucleotide repeats in the 3' untranslated region of the DMPK gene. Although patient myoblasts have been used for studying the disease in vitro, the invasiveness as well as the low accessibility to muscle biopsies motivate the development of alternative reliable myogenic models. Here, we established two DM1 induced pluripotent stem (iPS) cell lines from patient-derived fibroblasts and, using the PAX7 conditional expression system, differentiated these into myogenic progenitors and, subsequently, terminally differentiated myotubes. Both DM1 myogenic progenitors and myotubes were found to express the intranuclear RNA foci exhibiting sequestration of MBNL1. Moreover, we found the DM1-related mis-splicing, namely BIN1 exon 11 in DM1 myotubes. We used this model to test a specific therapy, antisense oligonucleotide treatment, and found that this efficiently abolished RNA foci and rescued BIN1 mis-splicing in DM1 iPS cell-derived myotubes. Together, our results demonstrate that myotubes derived from DM1 iPS cells recapitulate the critical molecular features of DM1 and are sensitive to antisense oligonucleotide treatment, confirming that these cells can be used for in vitro disease modeling and candidate drug testing or screening.This article has an associated First Person interview with the first author of the paper.

Keywords: Induced pluripotent stem (iPS) cells; Muscular dystrophy; Myotonic dystrophy; PAX7; RNA foci; Skeletal myogenesis.

Fig. 1.

Molecular characterization of DM1 patient-derived fibroblasts and reprogrammed DM1 iPS cells. (A) Southern blot analysis using a digoxigenin-labeled probe binding to the 3′ UTR of the DMPK gene to determine the length of CTG repeats in fibroblast samples from two DM1 patients (referred to as DM1-1 and DM1-2). Fibroblasts from an unaffected individual were used as control. The DM1-1 sample showed an expansion of ∼2000 CTG repeats, whereas the DM1-2 sample contained ∼1500 CTG repeats. (B) Representative RNA-FISH images showing foci only in fibroblasts from DM1-1 and DM1-2 patients. A Cy3-labeled (CAG)₇ probe was used to detect the foci (in red) and DAPI was used to stain nuclei (blue). Maximum projection of the Z sections is shown by confocal microscopy. Scale bar: 20 μm. (C) Quantification of foci (from B), represented as average number of foci per nucleus in 150 cells. Bars indicate s.d. from three independent experiments. (D) Representative images of RNA-FISH (red) coupled with immunostaining for the pluripotency marker Oct 3/4 (green) in DM1 iPS cells (iPSC) and control iPS cells (PSC). Maximum projection of the Z sections is shown by confocal microscopy. Scale bar: 20 μm. (E) Quantification of foci (from D), represented as average number of foci per nucleus in 150 cells. Bars indicate s.d. from three independent experiments.

Fig. 2.

Characterization of DM1 myogenic progenitors. (A) Representative images showing RNA-FISH of foci (red) co-stained with the myogenic transcription factor PAX7 (green) in myogenic progenitors derived from control and DM1 iPS cells using confocal microscopy. Maximum projection of the Z sections is shown. Scale bar: 20 μm. (B) Quantification of foci (from A), represented as average number of foci per nuclei in 150 cells. Bars represent s.d. from three independent experiments. (C) Representative images showing RNA-FISH of foci (red) co-stained with the splicing factor MBNL1 (green) in myogenic progenitors derived from control and DM1 iPS cells. Mid Z section is shown. Scale bar: 20 μm.

Fig. 3.

Terminal differentiation of DM1 patient-specific iPS cell-derived myogenic progenitors into myotubes. (A) Representative images showing RNA-FISH of foci (red) co-stained with MYHC (green) in control- and DM1-derived myotubes. Confocal microscopy shows maximum projection of the Z sections. Scale bar: 10 μm. (B) Quantification of foci (from A), represented as average number of foci per nuclei in 150 cells. Bars represent s.d. from three independent experiments. (C) Representative images of RNA-FISH (red) coupled with immunostaining of the splicing factor MBNL1 (green) in control, DM1-1 or DM1-2 myotubes, analyzed by confocal microscopy. Mid Z section is shown. Scale bar: 20 μm.

Fig. 4.

Antisense oligonucleotide treatment reverses the molecular phenotype of DM1 iPS cell-derived myotubes. (A) Representative images showing RNA-FISH of foci (red) coupled with immunostaining of MYHC (green) following 2′-OMe-PT-(CAG)7 antisense oligonucleotide treatment on DM1-1 or DM1-2 myotubes. Scale bar: 20 μm. (B) Number of nuclei showing RNA foci in three independent experiments (n=100). Data are mean±s.e.m. Comparisons were performed using the Mann–Whitney test. (C) Reverse transcription polymerase chain reaction (RT-PCR) analysis of BIN1 exon 11 following 2′-OMe-PT-(CAG)7 antisense oligonucleotide treatment in control, DM1-1 or DM1-2 differentiated myotubes. (D) Percentage of BIN1 exon 11 inclusion (from C) from three independent replicates. Data are mean±s.e.m. Comparisons were performed using the Mann–Whitney test. ***P<0.001.