Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro

Abstract

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Figure 1. Generation of MyoD-hiPSCs.

(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.