Figure 10.

Transmission electron microscopic (TEM) analysis of CCCs (hMSCs culture, hiPSC-ECMs culture, and hiPSC-ECMs/hMSCs co-cultures). hMSCs cultured on CCCs in myocyte medium showed euchromatic nuclei (N) with single to multiple nucleoli (n), and the cytoplasm was characterized by numerous random mitochondria and widespread endoplasmic reticulum, the ultrastructural characteristics of an active and immature cells (A). Besides, the hMSCs cytoskeleton exhibited bundles of actin stress fibers (sf) predominantly confined toward the peripheral cytoplasm. When hiPSC-ECMs cultured on CCCs under the same culture conditions revealed active euchromatic nucleus (N). The cytoplasm revealed myofibrils (mf), perinuclear early sarcomeric units, Z-discs (Z), and few mitochondria (mi) interspersed between the myofibrils along with glycogen particles (B). Whereas, the hiPSC-ECMs/hMSCs CCC co-cultures revealed that the differentiating hMSCs were in juxtaposition and tethered by numerous focal adhesions localized along the cellular margins of adjacent hiPSC-ECMs. The myofibrils of the coupled myocytes frequently exhibited a number of discontinuous and thickened Z discs as well as indistinct or absent M bands. Disorganized actin and myosin filaments not assembled into myofibrils were also seen in the cytoplasm (C). Some disorganized thin filaments were continuous with myofibrils, and were often attached to the sarcolemma by focal adhesions (C). Occasional developing myocytes illustrated typical imperfections of the myofibrillar organization, characterized by discontinuous and/or widened Z discs, indistinct H zones, free floating myosin filaments, as well as large number of glycogen granules; apparently mimicking like a specialized sino-atrial type of cells; besides, there were evidently numerous ovoid or circular electron-dense bodies, the Z-bodies (Zbs), composed of short sarcomeric units of α-actinin, the Z-bands were created by the fusion of these precursor Z-bodies (D). In addition, some differentiating myocytes showed branching and strand-like myofibrils with regular Z disks and distinct A and I bands. The myofibrils were stretched through the entire length of the cells, with scattered pleomorphic mitochondria and tubules of sarcoplasmic reticulum. Glycogen granules were also widespread throughout the remainder of the cell. These myocytes were appeared to be fused with their adjacent hMSCs, with imperceptible demarcation of their cellular boundaries (E,F). (A,C,D,F, scale bar 2 μm; B, scale bar 500 nm; E, scale bar 10 μm).