Human myotube differentiation in vitro in different culture conditions

Abstract

Human muscle cells derived from satellite cells, maintained in standard tissue culture conditions, do not differentiate as rapidly or as completely as myoblasts from other species (chicken, rat, mouse). In an attempt to improve myogenesis, we studied the effects of modifying the culture media and of coculturing muscle with nerve cells, using myoblasts grown in standard culture media as the basis for comparison. Myogenesis was measured by fusion index, creatine kinase (CK) activity; acetylcholinesterase (AChE) activity (total and molecular forms); and the number of acetylcholine receptors (AChR). Modification of culture media accelerated fusion of myoblasts, but the cell density decreased and myotubes were unable to survive for long periods. In contrast, coculturing muscle with nerve cells increased both cell density and the number of myotubes. CK, AChE and AChR increased in the presence of defined media. In the nerve-muscle cocultures the increase was less marked. Manipulating culture conditions modified the molecular forms of AChE. Only a (4 + 6.5) S peak was present in control cultures, but a 10S peak appeared in defined media. The 16S form was detected only in nerve-muscle cocultures. This study shows that fusion of human myoblasts and differentiation of myotubes in tissue culture can be accelerated by removal of serum macromolecules. Further differentiation of myotubes was achieved only in the nerve-muscles cocultures.