Degree of Differentiation

Maturation of cultured myotubes can be studied by various methods. The morphology changes dramatically during development (Figure 3). Early myotubes are relatively thin and short with centrally located, clustered nuclei. More differentiated myotubes are longer and thicker, and nuclei are often more peripheral. With phase-contrast, development of cables (immature myofibrils) can be seen, which occupy most of the sarcoplasm, forcing the nuclei to the subsarcolemmal region. From these immature myofibrils, structures with an obvious cross-striated appearance develop. Eventually, these align and the whole, often spontaneously contracting, myotube displays a myofibrillar cross-striation.

This process can be studied immunocytochemically using antibodies recognizing components such as titin (Figure 4) [52, 85, 99]. The supramolecular organization of this giant protein is a good marker for the development of myofibrils and thus, the differentiation of myotubes [52]. In proliferating myoblasts titin cannot be detected, but in differentiating human skeletal muscle cells, titin is one of the first myofibrillar proteins expressed after the induction of differentiation, even before cell elongation or cell fusion occurs. In these cells, titin is distributed in a punctate pattern. Then these titin aggregates associate with actin bundles, the so-called stress fiberlike structures. During further differentiation, titin is reorganized in longitudinal fibrils that eventually change to cross-striated myofibrils. Terminal differentiation is reached when the previously individual myofibrils lie in register and the myotubes show a mature cross-striated morphology when stained for titin [52, 99].

Biochemical assays include analysis of creatine kinase (CK) isozymes. CK is composed of two subunits (CK-B and/or CK-M). In proliferating myoblasts, only the CK-BB isoform is detected, and in differentiated normal adult muscle, the muscle CK-MM is almost the only isoform present. Intermediate stages contain a mixture of both isoforms together with the heterodimer CK-MB. The assays take advantage of the fact that during embryogenesis, regeneration and in vitro differentiation, the levels of the individual CK dimers present in the muscle cells gradually change [ 114, 136-138]. The percentage of CK-MM is a marker for the maturation grade. The highest percentages of CK-MM reported in aneurally cultured human myotubes and using optimized media were over 60% [51, 139], while in cultures with standard culture media, CK-MM percentages of 15 to 20% are usually reached [51]. The activity of CK and other enzymes such as cytochrome c oxidase, phosphorylase, citrate synthase and AMP deaminase also correlate with the maturation grade, and are useful differentiation markers [51, 89].

Figure 3. Assembly of myofibrils in phase contrast photomicrographs of methanol-fixed cultured human skeletal muscle cells during several stages of differentiation. In proliferating cell cultures (A), cells are mononuclear and mitotic cells with separated sister chromatids may be observed. Shortly after induction of differentiation, the first myoblasts fuse to form small myotubes (B). In these developmental stages, hardly any filamentous material is observed. During the next few days, mononuclear myoblasts fuse with the early myotubes and large, multinuclear myotubes are observed (C-J). The amount of filamentous material gradually increases. During the first approximately two days of differentiation, this material has a thin and non-structured appearance (C-E), but occupies most of the myotube, forcing the nuclei to the periphery. Thick cables later appear (F) that gradually develop cross-striations (F,G), indicating that myofibrillar structures are assembled (2 - 4 days of differentiation). The sarcoplasm of the most mature myotubes, after approximately 6 days of differentiation (H,J), is filled with large numbers of fully developed myofibrils with discernable A-bands, I-bands, and Z-discs (arrows in J, inset). Bar: 20 |xm or 8 |xm (inset).

Figure 3. Assembly of myofibrils in phase contrast photomicrographs of methanol-fixed cultured human skeletal muscle cells during several stages of differentiation. In proliferating cell cultures (A), cells are mononuclear and mitotic cells with separated sister chromatids may be observed. Shortly after induction of differentiation, the first myoblasts fuse to form small myotubes (B). In these developmental stages, hardly any filamentous material is observed. During the next few days, mononuclear myoblasts fuse with the early myotubes and large, multinuclear myotubes are observed (C-J). The amount of filamentous material gradually increases. During the first approximately two days of differentiation, this material has a thin and non-structured appearance (C-E), but occupies most of the myotube, forcing the nuclei to the periphery. Thick cables later appear (F) that gradually develop cross-striations (F,G), indicating that myofibrillar structures are assembled (2 - 4 days of differentiation). The sarcoplasm of the most mature myotubes, after approximately 6 days of differentiation (H,J), is filled with large numbers of fully developed myofibrils with discernable A-bands, I-bands, and Z-discs (arrows in J, inset). Bar: 20 |xm or 8 |xm (inset).

Figure 4. Immunocytochemical analysis of myofibril assembly. Depicted are immunofluorescence micrographs of different stages of methanol/acetone fixed proliferating (A) or differentiating (B-F) human skeletal muscle cells stained with an antibody that recognizes a titin epitope close to the Z-disc. Whereas in proliferating myoblasts titin cannot be detected (A), within 16 hours following the induction of differentiation, titin is expressed in a diffuse or punctate pattern (B). Subsequently, these titin aggregates associate with actin bundles, the so-called stress fiber-like structures, resulting in a continuous staining of these structures (C). During further differentiation, titin is reorganized and observed as regularly spaced dots (D) that eventually change into cross-striations (E). In mature myotubes, individual myofibrils register and the complete myotube shows a cross-striated morphology when stained for titin (F). Bar: 25 |xm.

Figure 4. Immunocytochemical analysis of myofibril assembly. Depicted are immunofluorescence micrographs of different stages of methanol/acetone fixed proliferating (A) or differentiating (B-F) human skeletal muscle cells stained with an antibody that recognizes a titin epitope close to the Z-disc. Whereas in proliferating myoblasts titin cannot be detected (A), within 16 hours following the induction of differentiation, titin is expressed in a diffuse or punctate pattern (B). Subsequently, these titin aggregates associate with actin bundles, the so-called stress fiber-like structures, resulting in a continuous staining of these structures (C). During further differentiation, titin is reorganized and observed as regularly spaced dots (D) that eventually change into cross-striations (E). In mature myotubes, individual myofibrils register and the complete myotube shows a cross-striated morphology when stained for titin (F). Bar: 25 |xm.

Electrophysiological methods to estimate maturity of muscle cultures include the analysis of resting membrane potentials [95, 140-142]. Dependent on the differentiation grade of the myotubes, membrane potentials may achieve values close or equal to those of around -60 to -80 mV reported for adult human muscle [140, 143].

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