Figure 1.
(a) Construction of the Tet-inducible MYOD1 expressing piggyBac vector (Tet-MyoD1 vector). (b) A scheme of generation of MyoD-hiPSCs. Human iPSCs were transfected the Tet-MyoD1 vectors with transposase by lipofection. To select transfected cells, G418 were added for 5 days in the hiPSC culture media at 2 days after transfection. (c) MyoD-hiPSCs after 24 h in culture with or without Dox administration. Scale bar = 20 µm. (d) Upper lanes show dox-added MyoD-hiPSCs at d1. Lower lanes show immunodetection of MHC in Dox-induced MyoD-hiPSCs at d7. A lower-left panel shows the cells differentiated in maintenance medium from d1 to d7. A lower-right panel shows the cells differentiated in αMEM containing 5% KSR from d1 to d7. Scale bars = 200 µm. (e) Percentage of MHC positive cells per total cells following MyoD-induced differentiation. **p<0.01.
Figure 2.
Optimization of Differentiation Conditions.
(a) Percentage of MHC positive myogenic cells derived from MyoD-hiPSCs during 9 days differentiation with various timing of medium replacement. *p<0.05 (b) The average number of nuclei of myofibers in each condition of 5% KSR or 5% HS containing media after 7 days differentiation. (c) Flow cytometric analysis of MyoD-hiPSCs with 24 h Dox treatment in different start points. Dox addition at differentiation d1 promoted higher percentage of mCherry expression in MyoD-hiPSCs than Dox addition at differentiation d4. (d) A merged image of phase-contrast and mCherry images in differentiated MyoD-hiPSCs which were administrated Dox at differentiation d4. Some MyoD-hiPSCs turned to be unresponsive with Dox, indicating no mCherry expression area (dotted line). Scale bar = 100 µm. (e) MHC positive myogenic cell number derived from MyoD-hiPSCs during 11 days differentiation with various administration periods of Dox. *p<0.05.
Figure 3.
Reproducible myogenic differentiation with the optimized protocol.
(a) A schematic of our muscle differentiation protocol beginning with MyoD-hiPSCs. (b) Immunohistochemistry of differentiated MyoD-hiPSCs for MHC (red). Scale bar = 100 µm. (c) Percentage of MHC positive cells per total cells following MyoD-induced differentiation of 6 MyoD-hiPSC clones. (n = 3 for each clone). Data are listed as mean ±S.D.
Figure 4.
Characterization of myofiber derived from MyoD-hiPSCs.
(a) Time course gene expression profile for undifferentiated and myogenic markers in B7 #9 MyoD-hiPSC clone with (gray bars) or without (black bars) Dox administration (n = 3). Data are listed as mean±S.D. The data were standardized by β-actin using teratoma. The data on d0 = 1 in undifferentiated markers, such as OCT3/4, SOX2 and NANOG. The data on d7 = 1 in other analyses. *: p<0.05, **: p<0.01, respectively, between Dox(–) and Dox(+). (b) Intracellular localization of mitochondria in both undifferentiated and differentiated MyoD-hiPSCs. Scale bar = 20 µm. (c) Immunohistochemistry of differentiated MyoD-hiPSCs for mature myogenic markers, such as CK-M, creatine kinase muscle isoform, Skeletal muscle Actin, and DYSTROPHIN. Scale bar = 20 µm. (d) Heat map of global mRNA expression comparing undifferentiated hiPSC (sample 1) and differentiated myogenic cells (samples 2-5). (e) Myogenic gene profile and unsupervised clustering based on markers associated with myofibers for undifferentiated hiPSCs and differentiated myogenic cells. Red color indicates up-regulated genes and blue color indicates down-regulated genes in (d) and (e).
Figure 5.
Functional assay for differentiated MyoD-hiPSCs.
(a, b) Electron microscopy of differentiated MyoD-hiPSCs (a) and differentiated human myoblast Hu5/E18 cells (b). Red arrows indicate myofibrils. Black arrowheads indicate future Z lines. Black arrows indicate myosin fibers. Scale bars = 500 nm. (c) Serial photographs of differentiated MyoD-hiPSCs co-cultured with C2C12 cells. A hiPSC-derived mCherry+ cell (white arrow) fused with a mouse-derived GFP+ cell (white arrowhead) resulting in a yellow cell (red arrow). Time increments between images = TIME. Scale bar = 100 µm. (d) Immunohistochemistry of MyoD-hiPSCs co-cultured with C2C12 cells. White arrows indicate human nuclei in a GFP+ murine myofiber. Scale bar = 100 µm.
Figure 6.
Modeling Miyoshi Myopathy (MM) by patient derived-hiPSCs.
(a) Morphology of patient derived MM-hiPSC clones, expanded following G418 selection for Tet-MyoD1 vector transposition. Scale bar = 200 µm. (b) RT-PCR analysis of endogenous pluripotent stem cell markers in MyoD-MM hiPSCs. (c) Efficient myogenic differentiation of MyoD-MM hiPSCs according to the protocol defined in Figure 3a. MHC positive (left), or Myogenin positive (right) cells were observed dominantly. Scale bars = 100 µm. (d) DYSFERLIN expression of the myofibers from MyoD-MM hiPSCs (lane 1, 2), rescued MyoD-MM hiPSCs which expressed full-length DYSF cDNA driven by EF1α promoter (lane 3, 4), and control non-diseased MyoD-hiPSCs (lane 5) confirmed by western blotting. ACTB = β-actin. (e) Entry of FM1-43 green fluorescent dye into differentiated myofibers from MyoD-MM #5 (left), rescued MyoD-MM #5 with DYSF expression (middle), or control MyoD-hiPSC clone B7 #9 (right), before (0 s) and 20 s after (20 s) two photon laser-induced damage of the sarcolemmal membrane (arrow). Scale bars = 20 µm. (f) Summary time course data of accumulation of FM1-43 dye in laser-damaged myofibers derived from B7 #9 (black circles), MyoD-MM hiPSCs (red or blue triangles) and rescued MyoD-hiPSCs with DYSFERLIN expression (red or blue circles). n = 5 for each clone. Data are listed as mean ±S.E.