Skip to main content
Fig. 1 | Skeletal Muscle

Fig. 1

From: Acute conversion of patient-derived Duchenne muscular dystrophy iPSC into myotubes reveals constitutive and inducible over-activation of TGFβ-dependent pro-fibrotic signaling

Fig. 1

Generation of stable hESC and iPSC lines and myogenic differentiation protocol. a Top: Scheme of inducible MyoD and Baf60c expression vectors generated using the enhanced version of piggyback (ePB) as described [16]. Puro, puromycin resistance; Bsd, blasticidin resistance; UbcP, ubiquitin C constitutive promoter; TRE, Tet-responsive element. Bottom: Protocol for myogenic differentiation of puromycin and blasticidin-resistant cells (hESC or iPSCBM) obtained after stable integration of the ePB vectors. Differentiation protocol starting from hESCBM and iPSCBM. Transgene expression is achieved by the addition of doxycycline (doxy, red line) in the hES medium for 24 h. Myogenic conversion is then triggered by switch to the proliferation medium (GM) at day 1 and to the differentiation medium (DM) at day 3. b Representative immunofluorescence images of hESCBM or iPSCBM at each step of the protocol. iPSCBM at the pluripotent stages are marked by OCT4, followed by the expression of BAF60C (green) and MyoD (red) upon doxy induction at d1 and the appearance of MYOGENIN at d3. At day 7, myotubes are visible and express the marker of terminal differentiation myosin heavy chain (MYHC, green) containing MYOGENIN-positive nuclei (Myog, red). Nuclei are counterstained with DAPI. Scale bar, 50 μm. c Relative gene expression by qRT-PCR of the indicated genes in a time course. Data are represented as average ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t test). d Quantification of markers of myogenic conversion efficiency (e.g., MyoD, Myog, MyHC) in MyoD/BAF60C expressing hESC and hiPSC after 7 days of culture in DM (as average ± SEM)

Back to article page