Generation and characterization of control and DMD patient-derived Tet-MyoD-transfected hiPS cells.
(a) Construction of the tetracycline-inducible mCherry-linked MyoD piggyBac vector (Tet-MyoD). (b) RT-PCR analysis of Control-iPStet-MyoD (Father and B7) and DMD-iPStet-MyoD (Δ44 and Δ46–47) for pluripotency markers (Oct3/4, Sox2 and Nanog). (c) Immunocytochemistry for SSEA4 and TRA1–60 and alkaline phosphatase staining demonstrated the pluripotent state of Control-iPStet-MyoD Father and DMD-iPStet-MyoD Δ44. Scale bar, 200 μm. (d) Skeletal muscle induction scheme for Tet-MyoD-transfected hiPS cells. Differentiation was initiated with Dox addition at day 1. Cells were cultured with 20% knockout serum replacement (KSR) hiPS medium for the first 2 days. (e) Quantitative RT-PCR analysis of Control-iPStet-MyoD and DMD-iPStet-MyoD showing relative expression of pluripotency markers Oct3/4, Nanog and Sox2 and (f) myogenic markers exogenous- and endogenous-MyoD and Myogenin during the differentiation process. Data were normalised by setting day 0 = 1 for pluripotency markers, day 2 = 1 for exogenous-MyoD and day 9 = 1 for endogenous-MyoD and Myogenin. n = 3. Beta-actin was used as an internal control. (g) Relative expression of endogenous-MyoD and Myogenin at day 9 of differentiation in each sample. Gene expression levels were normalised by setting levels in the human myoblast cell line Hu5/E18 at day 5 of differentiation = 1. Ubiquitin C was used as an internal control. Black and white bars indicate Control- and DMD-Myocytes, respectively.