Fig 2: Effects of miRNAs on myogenic differentiation.a, b Expression of myogenic differentiation marker genes. MiRNAs (indicated) that are more abundant in young mouse blood than in aged mouse blood were examined. Synthetic miRNAs (Mission® microRNA mimics) were introduced into C2C12 cells; and 2 and 3 days after miRNA introduction, the expression of Myog (a) and Myh1 (b) was examined by qRT-PCR as in Fig. 1b. Cells treated with non-silencing control RNAs (nsCont) and untreated cells (-) were also examined as a control. Normalized data were further normalized with the data obtained from cells treated with nsCont as 1. Data are shown as mean ± SEM (n = 3 independent determinations; *p < 0.001 by Dunnett’s test). c Myosin heavy chain (MyHC) expression. C2C12 cells transfected with Mission miR-199-3p mimic [199(HMI0338)] and nsCont were cultured for 4 days. Cell lysate was prepared and examined by western blotting using antibodies against MyHC and a-tubulin (as a loading control).

Fig 3: The model of N1ICD overexpressed PSCs. (A) The expression of N1ICD was tested by immunocytochemistry. DAPI, blue, represents nuclei; NOTCH1, red; Merge, pink, represents N1ICD expressed in nuclei of PSCs. (B) One week later, the degree of green fluorescence protein (GFP) in the control group is greener than the N1ICD-overexpressed group. However, the level of N1ICD expression in the N1ICD-overexpressed group is more than the control group. (C,D) Representative images of the immunofluorescent staining for proliferating PSCs are shown. Proliferating PSCs were labeled with Edu fluorescent dye (red). (E) Q-RT-PCR showed the changes of N1ICD, hes family bHLH transcription factor 5 (HES5), paired box 7 (PAX7), myogenic differentiation 1 (MYOD), cyclin dependent kinase inhibitor 1A (P21) and cyclin D1 (CCND1) in proliferating PSCs. Overexpressed N1ICD in PSCs did not show any changes in mRNA level of MYOD and CCND1. * p < 0.05; ** p < 0.01. Data are the mean ± S.E.M, n = 3 for each treatment. (F) Representative images of the immunofluorescent staining for differentiating PSCs are shown. Myosin heavy chain (MYHC) was labeled by fluorescent dye (red). Scale bar = 20 µm (×200 magnification). (G) Q-RT-PCR showed the changes of myogenin (MYOG), PAX7, MYOD and MYHC in overexpressed N1ICD differentiating PSCs. MYOG and MYOD significantly decreased while PAX7 significantly increased in overexpressed N1ICD differentiating PSCs. * p < 0.05; ** p < 0.01. Data are the mean ± S.E.M, n = 3 for each treatment.

Fig 4: Motor neuron-myocyte culture on aligned nanofibers.a, b C2C12 Skeletal myocytes and spinal motor neurons were cultured separately as monocultures on aligned PCL nanofiber sheets to optimize media conditions and subsequently stained with Phalloidin and Tuj-1, respectively. Scale bar = 200 µm. c Subsequently, myocytes and motor neurons were cocultured on nanofiber sheets for optimizing coculture media. Scale bar = 200 µm. d In order, to confirm the formation of myotubes and neuromuscular bundles the motor neuron-myocyte cocultures on nanofiber sheets were stained for Myosin Heavy Chain (MHC) and Tuj-1. The neuromuscular bundles were visualized from compressed multiple Z-stack images in planar as well as volumetric view. Scale bar = 100 µm (Planar view), 70 µm (Volume view). The planar and volume view are from the same culture.

Fig 5: Generation of DM1-iPSCs and their differentiation.(A) The strategy of our study: patient iPSCs were passed and differentiated at three different passage numbers into CMs or neurons giving 9 samples (left), or had a MyoD1 vector transfected and were differentiated into myocytes, giving 6 samples (right). The CTG repeat lengths were measured in each sample. (B) Six clones from three different DM1 patients expressed pluripotent stem cell markers (Oct3/4, Nanog and Sox2) in conventional PCR. ß-actin was used as a loading control. (C) Karyotypic analysis of undifferentiated iPSCS (Pt-1B). (D, left) Representative live image of CMs on day 20 (Pt-1B). A video clip is available in Supplementary Video 1. (D, right) FACS analysis of the CMs shown in the picture on the left. The X-axis indicates the percentage of cardiac troponin T (cTnT)-positive cells among the total number of CMs. The Y-axis indicates the autofluorescence of the CMs. (E) Representative immunostaining image of neurons on day 42 (Pt-1B). The left panel shows neurons that expressed Tyrosine Hydroxylase (TH) and Microtubule-associated protein 2 (Map2). The right panel shows neurons that expressed TH and Neuron-specific Class III ß-tubulin (TUJ1). (F) Representative immunostaining image of myocytes on day 7 (Pt-1B). The myocytes expressed Myosin Heavy Chain (MHC). Hoechst stains the nuclei.