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Additional file 1: Figure S1. Fiber type composition of myotubes resulting from the in vitro differentiation of PSC-derived iPax3/iPax7 myogenic progenitors and adult satellite cells. Representative images show immunofluorescence staining for pan, embryonic, type I and IIA MyHC isoforms (red) in myotubes resulting from the in vitro differentiation of iPax3 and iPax7 PSC-derived myogenic progenitors, as well as adult satellite cells (adult). Nuclei were counterstained with DAPI (blue). Magnification bar: 100 μm. Figure S2. Characterization of fiber type composition. a) Graph shows the ratio of oxidative DYS+MyHC+ type I and type IIA over glycolytic type IIX and IIB myofibers following the transplantation with iPax3 and iPax7 myogenic progenitors, and adult satellite cells. Data are shown as mean ± SEM (n = 7-8 per group). **p < 0.01. b) Graph bars show percentage of DYS+MyHC+ double positive with respect to total DYS+ in TA muscles transplanted with satellite cells isolated from TA or soleus muscles. Data are shown as mean ± SEM (n = 4 per group) and no statistically significant differences were observed among groups. c) Representative images show staining for embryonic MyHC (red) and dystrophin (green) in TA muscles from NSGmdx4cv mice that had been injected with iPax3 or iPax7 myogenic progenitors. Adult satellite cells served as control. Magnification bar: 100 μm. d) Graph shows respective quantification (panel c), as indicated by the number of DYS+MyHC+ double positive with respect to total DYS+. Data are shown as mean ± SEM (n = 6-8 per group). *p < 0.05 and ***p < 0.001. d) Quantification of CSA (μm2) of grafts from indicated groups. Data are shown as mean ± SEM (n = 4-5 per group). *p < 0.05. Figure S3. Characterization of fiber type composition following the transplantation of embryonic, fetal, and neonatal primary cells. a) Representative images show staining for MyHC isoforms (red) and dystrophin (green). Nuclei were counterstained with DAPI (blue). Magnification bar: 100 μm. b) Percentage quantification of DYS+MyHC+ double positive with respect to total donor-derived DYS+ myofibers. Data are shown as mean ± SEM (n = 4-5 per group) **p < 0.01. Figure S4. Characterization of fiber type composition based on recipient genetic background. a) Representative images show staining for type I MyHC (red) and Laminin (green) in TA muscles from dystrophic NSGmdx4cv and NSG mice. b) Graph shows respective quantification (panel a), as indicated by the number of MyHC+ myofibers in TA muscles from dystrophic NSGmdx4cv and NSG mice. Data are shown as mean ± SEM (n = 6 per group). *p < 0.05, **p < 0.01, and ***p < 0.001. c) Graph bar shows percentage of MyHC+ donor-derived myofibers from Fig. 2a in comparison to iPax3 and iPax7 samples from Fig. 1b. Data are shown as mean ± SEM (n = 6-8 per group), and no statistically significant differences were observed among groups. Figure S5. Characterization of fiber types from secondary grafts. a) Representative image shows staining for embryonic MyHC (red) and dystrophin (green) in TA muscles of secondary recipients that had been injected with iPax3-derived MNCs. Magnification bar: 100 μm. b) Graph shows respective quantification (panel a), as indicated by the number of DYS+MyHC+ double positive with respect to total DYS+ myofibers of secondary grafts (iPax3 MNCs) in comparison to primary-injected TAs (iPax3). Data are shown as mean ± SEM (n = 4 per group). **p < 0.01.

Additional file 1: Supplementary Figure S1. Histological characterization of FKRPP448L-NSG mice. a) Representative images show H&E staining in TA muscle cryosections from 7-week-old B6 (control) and FKRPP448L-NSG mice. Arrows indicate centrally located nuclei and asterisks denote the presence of infiltrating mononuclear cells. Scale bar is 50 μm. b) Quantification of the percentage of centrally nucleated myofibers in the TA muscles of 7-weel-old B6 and FKRPP448L-NSG mice. Data are shown as mean + SEM (TA muscles from 4 mice). ***p < 0.001. Supplementary Figure S2. Mouse ES cell labelling/differentiation and human iPS cell differentiation. a) Outline representing the labeling of iPax3-GFP mouse ES cells with the H2B-RFP encoding lentivirus and subsequent myogenic differentiation. b) Representative FACS plots show percentage of RFP+ cells at different stages of differentiation: left: ES cells, center: embryoid bodies (EBs) before sorting, and right: myogenic progenitors used for transplantation (P4). c) Representative images show immunostaining for IIH6 and RFP in myotubes resulting from the in vitro differentiation of ES cells. IIH6, RFP, and nuclei are shown in green, red and blue, respectively. Scale bar 50 μm. d) Outline representing the timeline of myogenic differentiation of human iPAX7 iPS cells. Supplementary Figure S3. Characterization of human engraftment. a) Representative images show immunostaining for human DYSTROPHIN (in gray) and human LAMIN A/C (in red) in muscle sections from CTX-injured FKRPP448L-NSG mouse TA muscles that had been injected with human iPS cell-derived myogenic progenitors or PBS (from Fig. 2c). DAPI stained nuclei (in blue). Scale bar is 100 μm. b) Representative images show satellite cell staining in the TA muscles described in (a). Circles show cells double-positive for PAX7 (green) and LAMIN A/C (red) under the basal lamina (Lam in gray) indicating donor-derived satellite cells. Nuclei in blue. Scale bar is 50 μm. c) High magnification image of donor-derived satellite cell. Scale bar is 20 μm. Supplementary Figure S4. Engraftment analysis in non-injured muscles of FKRPP448L immunocompetent mice. a) Representative images show immunostaining for IIH6 (in green) and RFP (in red) in non-injured TA muscles from FKRPP448L mice that had been injected with PBS (upper panel) or mouse ES cell-derived myogenic progenitors (lower panel). DAPI stained nuclei (in blue). Scale bar is 100 μm. b) Engraftment quantification based on the number of RFP+/IIH6+ myofibers (from a). Data are shown as mean + SEM (n = 5; 2 males and 3 females). c) Distribution of the number of RFP+/IIH6+ myofibers along the TA muscle (n = 5; 2 males and 3 females). Supplementary Figure S5. Engrafted area quantification in non-injured muscles of FKRPP448L-NSG mice. a) Representative image used to assess the size of the engrafted area (marked in red) compared to the total cryosection area (marked in blue). IIH6 (gray) and RFP (red) allow the delimitation of the area of engraftment. Scale bar is 500 μm. b) Distribution along the length of TA muscle of the percent engraftment (RFP+/IIH6+) area. Data are shown as mean + SEM (n = 7; 4 males and 3 females). Supplementary Figure S6. Engraftment analysis in non-injured muscles transplanted with human iPS cells. a) Representative images show immunostaining for IIH6 (in green) and human LAMIN A/C (in red) in muscle sections from non-injured FKRPP448L-NSG mouse TA muscles that had been injected with human iPS cell-derived myogenic progenitors (lower panel) or PBS (upper panel). DAPI stained nuclei (in blue). Scale bar is 50 μm. b) Engraftment quantification based on the number of IIH6+/LAMIN A/C+ myofibers (from a). Data are shown as mean + SEM (n = 6, 4 males and 2 females). c) Distribution of the number of IIH6+/LAMIN A/C+ myofibers along the TA muscle (n = 6; 4 males and 2 females). d) Representative images show immunostaining for human DYSTROPHIN (in gray) and human LAMIN A/C (in red) in muscle sections from non-injured FKRPP448L-NSG mouse TA muscles injected with iPS cell-derived myogenic progenitors or PBS (from a). DAPI stained nuclei (in blue). Scale bar is 50 μm. Supplementary Figure S7. Additional western blot analysis and Laminin overlay assay. a) Western blot for IIH6 and β-DG in TA lysates from 7-week-old FKRPP448L-NSG mice (2 TA muscles pooled) that had been injected at 3-weeks of age with mouse ES cell-derived myogenic progenitors. To determine the linear range of detection for IIH6 and β-DG antibodies, an increasing amount of protein (0, 25, 50, 100, 125, 150, 200 μg) was loaded. b) Quantification of IIH6 band intensity according to the amount of protein loaded. c) Quantification of the β-DG band intensity related to the amount of protein loaded. d) Western blot for IIH6 in TA lysates from 7-week-old FKRPP448L-NSG mice that had been injected at 3-weeks of age with mouse ES cell-derived myogenic progenitors or PBS (contralateral muscle as negative control). Data from two independent experiments (n = 5 for each), and their respective quantification of IIH6 band intensity normalized with β-DG. Data are shown as mean + SEM. *p < 0.01. e) Detection of laminin binding. No calcium served as negative control. f) Quantification of laminin band intensity normalized with β-DG. Data are shown as mean + SEM (n = 4; 2 males and 2 females). *p < 0.01.

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., Summary: This work provides proof of principle for the use of myotonic dystrophy 1 patient-specific induced pluripotent stem cells to model muscle pathology in vitro and in drug discovery.

To understand the effect of DUX4 in vivo, and to generate a model in which to test anti-DUX4 therapeutics, we have introduced a doxycycline (dox)-inducible transgene encoding DUX4 and 3′ genomic DNA into a euchromatic region of the mouse X chromosome. Although this mouse was intended to provide doxycycline-inducible phenotypes, through expression of DUX4, we found that the transgene alone, without dox, was lethal in males. Females were runted and presented a skin pathology in the absence of dox. This is due to low level leaky expression of the DUX4 gene, as transcript can be detected by PCR in most tissues with the most consistent detection in neural tissues including retina, and in testis. We could not detect the protein in the absence of dox. Although males are not usually born live, occasional males survive to term. These animals are much more severely affected than their female counterparts, and remarkably, display a retinal vascular pathology similar to that seen in FSHD. In addition, they show a defect in spermatogenesis. Muscles from these animals were composed of smaller fibers and were weaker, but proportionally so, and no dystrophy was present prior to death, invariably before 6 weeks of age. We performed ex vivo culture experiments with myoblasts and muscle fibroadipogenic progenitors (FAPs) isolated from the iDUX4 males, and found that high levels of dox caused cell death of myoblasts and was growth inhibitory to fibroblasts. Low non-toxic levels of dox impaired differentiation of myoblasts in vitro, but had no effect on adipogenic differentiation of muscle FAPs. To evaluate the effect of DUX4 expression during regenerative repair in vivo, we crossed the Pax7-ZsGreen satellite cell marker into this strain and performed transplants of satellite cells from live-born males into female NSG-mdx4Cv mice,and treated recipients with varying doses of dox. By using dystrophin staining to quantify engraftment, we observed a consistent and easily quantifiable dose-dependent reduction in regeneration by transplanted satellite cells. These data show that DUX4 expression, even at low levels, is inhibitory to regenerative myogenesis. We further propose that the quantification of donor-derived myofibers generated by transplanted iDUX4 satellite cells can serve as a useful in vivo test system for therapeutics directed against the DUX4 protein or transcript.

Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9-Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies