Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells

Abstract

The forced expression system of MYOD1, a master gene for myogenic differentiation, can efficiently and rapidly reproduce muscle differentiation of human induced pluripotent stem cells (hiPSCs). Despite these advantages of the MYOD1 overexpression system, developed myotubes are relatively immature and do not recapitulate several aspects of striated muscle fibers. Here, we developed a simple optimized protocol using an alternative culture medium for maximizing the advantages of the MYOD1 overexpression system, and successfully improved the formation of multinucleated mature myotubes within 10 days. In this study, we generated hiPSCs derived from healthy donors and an individual with congenial muscular dystrophy caused by LMNA mutation (laminopathy), and compared disease-associated phenotypes in differentiated myotubes generated by the conventional method and by our new optimized culture method. Using our optimized method, abnormal myonuclear shape was pronounced in the patient-derived iPSCs. In addition, abnormal accumulation of the nuclear membrane protein emerin was observed in LMNA-mutant hiPSCs. Our new culture method is expected to be widely applicable as a MYOD1 overexpression model of hiPSC-derived skeletal muscle cells for the analysis of a variety of muscle diseases.

Keywords: Differentiation of skeletal muscle cells; Induced pluripotent stem cells; Laminopathy; Muscular dystrophy; Newly developed culture method.

Fig. 1

Schematic diagrams of tested culture protocols using Healthy hiPSCs. (A) The standard protocol using 2%HS/DMEM after day 4 of culture. (B) A protocol using myotube medium on day 4. Cell culture became unsuccessful on day 10. (C) A protocol using fusion medium on day 4. Cell culture became unsuccessful on day 10. (D) A protocol using 2%HS/DMEM and myotube medium. (E) An optimized protocol using 2%HS/DMEM and fusion medium. (F) A protocol using myotube medium and fusion medium. Cell culture was failed at day 10. Y represents Y-27632 (ROCK inhibitor), and Dox represents doxycycline. Scale bar = 500 μm.

Fig. 2

Promotion of muscle maturation of Healthy, LMNA-mutant, and LMNA-rescue hiPSCs using the optimized protocol. (A) Representative immunohistochemical staining for MHC in the differentiated hiPSC lines cultured for 10 days with the standard or optimized protocol. Scale bar = 100 μm. (B) The myocyte induction efficacy calculated by the percentage of nuclei in the MHC-positive area. Five images (×20 objectives) were taken from each well of the three cell lines and analyzed. (C) Images of Western blots performed on three individual samples to quantify the levels of dystrophin, MHC, and actinin in the three cell lines. (D) Graph showing the relative expression of each protein to the levels of lamin B1 expression. (E) Frequency of myotubes with specific number of nuclei as an indicator of muscle maturation. Four to five images (×10 objectives) were taken from each well of the 3 cell lines and analyzed. (F) Quantification of genes associated with muscle differentiation by RT-qPCR. Relative mRNA levels of MYH3, MYH7, MYH2, ATP2A1, CASQ1, and TMEM8C were normalized to the level of TBP, and shown as the fold increase to cells cultured using the standard protocol. Data are shown as the mean ± SD (n = 3, each group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the standard protocol.

Fig. 3

Increased nuclear abnormalities in LMNA-mutant hiPSCs using the optimized protocol. (A) Immunohistochemical staining for MHC and lamin A, with a DAPI nuclear counterstain, for the detection of abnormalities in nuclear shape in Healthy, LMNA, and Rescue hiPSCs using the standard and optimized protocols. Scale bar = 100 μm. (B) Representative images of the abnormal shapes of nuclei stained with lamin A, characterized as blebs, mild deformation, elongation, stringing, and severe stringing using the optimized protocol. Each panel displays individual black and white images. Yellow arrows indicate representative nuclear abnormalities. Scale bar = 40 μm. (C) Graph showing percentages of myonuclei with abnormal shapes, and (D) graph showing average lengths of the major axis of nuclei within myotubes, calculated to evaluate myonuclear lengthening in the 3 hiPSC lines, cultured using either the standard or the optimized protocol. Data were obtained from 300 to 450 myonuclei of each cell line. Data are shown as the mean ± SD. ***P < 0.001.

Fig. 4

Localization of NE proteins in nuclei from differentiated myotubes of LMNA-mutant iPSCs. (A) Lamin B1, emerin, NUP153, and LAP2 were double-stained with lamin A/C and a DAPI nuclear counterstain. Yellow arrows indicate the representative localizations of nuclear envelope proteins in abnormal regions of myonuclei. (B) Accumulation of emerin expression in abnormal regions of myonuclei from LMNA hiPSCs cultured on a glass-bottom dish, a plastic-bottom dish, and a silicone chamber. Scale bar = 50 μm.