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Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients’ pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models.Graphical Abstract

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IntroductionIn recent years, the direct-bonded silicon on insulator (Sol) wafer has been considered promising for future device applications such as high-density integration and radiation-hardened substrates. Selective chemical mechanical polishing (CMP) with a mechanical stopper is a well known method for realizing ultra-thin SO1 layers[l-31. It is, however, difficult to produce large surface areas, because deformation from the polishing pad results in nonuniform SO1 layer thicknesses. In this paper, we produce a pH-controlled CMP technique for large-area ultra-thin SO1 layers with uniform thickness. In this technique, the grooves are fabricated on the SO1 layer to expose insulator oxide which functions as a chemical stopper producing a large-area, but uniformly thin, layer. When the pH of the polishing reagent is appropriately controlled, the silicon polishing rate drops spontaneously because a flocculating action occurs in the reagent's colloidal silica for reduced thickness. Figure 1 shows the fabrication sequence. We used thermally oxidized n-type 6-inch-diameter Si(lO0) wafers. The bonded SO1 wafers were made by pulse-field-assisted bonding [4]. Surface grinding and polishing enabled us to thin the SO1 layer to 3-5 pm. Experimental ProcedurePart of the SO1 layer was removed to expose the insulator oxide which flocculates the colloidal silica.The SCN layer was etched by KOH, leaving 5 by 5 mm Si islands. The width of the grooves between islands was 1 mm. Figure 2 shows the polishing method. The head with the SO1 wafer was oscillated across the length of the polishing pad, with both the head and the polishing pad revolving at 200 rpm. Polishing pressure was under 20 kPa and temperature was kept between 5°C and 15°C. A polyurethane pad and a diluted colloidal silica !jolution with amine (polishing reagent) were used. pH-controlled CMP was done with the polishing rea er3 concentration ranging from 0.01% to 0.06% (Si82W%). The pH of the reagent was controlled, in a range from 7 tolO, with hydrochloric acid (HCI) or ammonia solution (NH40H). Results and DiscussionExamining the SO1 layer thickness as function of reagent pH and polishing time (Fig. 3), the colloidal silica concentration was 0.06%. For a pH of 8.5, it is clear that the average polishing rate dropped according to the thickness and that the insulator oxide worked as a stopper. Due to the grooves, the average polishing rate was reduced to 0.007 pm/min from 0.05 pm/min. When the grooves were not present, the rate was unchanged even at pH of 8.5. For pH less than 7, polishing did not proceed because the colloidal silica, which existed only in the alkaline solution, is completely flocculated. A pH of 10 is equal to that of the conventional reagent and the average polishing rate was unchanged. Cross sectional SEM of the Si island after polishing (Fig. 4) shows that the SO1 layer surface is O.lpm higher than the insulator oxide surface.Since colloidal silica is easily flocculated for pH of 8 and due to acidity from the silanol groups on the surface of the i...