Fig 2: Differentiation of control and SJS-derived human-induced pluripotent stem cells (hiPSCs) into myotubular cells. (a) Myosin heavy chain (MHC) staining of myotubes derived from Con_#10, Con_#16, SJS_#10, and SJS_#15; pink shows MHC-positive cells and blue shows nuclei. (b) Differentiation index; the ratio of the number of nuclei in myotubes to the total number of nuclei. (c) The relative RNA expression levels of differentiation markers in skeletal muscle during differentiation. Mean and S.D. are indicated (n = 3 in each clone). As shown in Figure 1, D0, D2, D4, R2, and R4 correspond to days 0, 2, 4, 6, and 8 after the initiation of differentiation, respectively. UD: undetectable. Data were analyzed using a two-tailed Student’s t-test (b) and one-way ANOVA with Tukey’s multiple comparison (b, c). *: p < 0.05, **: p <0.01, ***: p < 0.001, ****: p < 0.0001. Scale bar, 100 µm.

Fig 3: Ca2+ influx in Schwartz–Jampel syndrome (SJS) patient-derived and control human-induced pluripotent stem cell (hiPSC) myotubes. (a) Pseudocolor images of F340/F380 under both resting conditions and peak activity in response to increasing acetylcholine (ACh) concentration (0, 0.03, 0.1, 0.3, and 1.0 µM) in control and SJS-hiPSC myotubes. (b) Representative traces of Ca2+ influx induced by ACh (0, 0.03, 0.1, 0.3, and 1.0 µM) in control and SJS-hiPSC myotubes (n = 5). (c) Dose-response relationship between Ca2+ influx and ACh in control (Con_#10 and #16) and SJS hiPSC myotubes (SJS_#10 and #15). Data were evaluated using the two-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (mean ± S.D., n = 5–6). a. p < 0.05 vs. Con_#10, b. p < 0.05 vs. Con_#16. Scale bar, 100 µm.

Fig 4: Growth-factor binding to rhPln.198, rhPln.247, and HSPG in solid phase. Commercial HSPG, Pln.247, or rhPln.198 were bound directly to polystyrene microtiter wells. The proteoglycan samples were incubated with 6-fold dilutions of rhFGF-2. After washing away unbound FGF, the bound FGF was detected with anti-FGF biotin conjugates.

Fig 5: Perlecan expression in myotubes derived from control and SJS human-induced pluripotent stem cells (hiPSCs). (a) Immunofluorescence analysis of perlecan derived from Con_#10, Con_#16, SJS_#10, and SJS_#15. Green and pink indicate domains III and IV, respectively. (b) The relative RNA expression levels of HSPG2 derived from hiPSCs. (c) The protein expression levels of perlecan extracellularly secreted from hiPSCs. Mean and S.D. are indicated (n = 3 in each clone). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparison (b,c). *: p < 0.05, ***: p < 0.001, ****: p < 0.0001. Scale bar, 100 µm.