A cellular model system of differentiated human myotubes

Abstract

The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc-cultures. In order to identify the differentiation conditions which give a good survival of myotubes and a high grade of differentiation, Sc-cultures were induced to differentiate in media supplemented with either 2% fetal calf serum (FCS) 2% horse serum (HS) or 10% HS. Based on higher CK-activities in cultures differentiating in FCS-supplemented media compared to horse sera, fetal calf serum was chosen to induce differentiation. The ATP, DNA and protein content increased during the first 4 days after induction of differentiation and was followed by a period with minor changes. The maximal differences of ATP, DNA and protein between days 4-10 were evaluated and the differences in the three components were found to be less than 20% of the average value with a certainity of more than 0.9. Day 8-myotubes were investigated morphologically and were found immunoreactive for fast myosin, and expressed areas with clear cross striation. We recommend the use of differentiated Sc-cultures in the period from day 4 to 8 after induction of differentiation as only minor differentation-related changes will take place in the cells during this period of time.

Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells

“…Cell culture procedures and 2-DG uptake Human cell cultures from lean individuals were established as described previously [19][20][21] (Table 1). Cells were cultured in DMEM supplemented with 2% (vol./vol.)…”

“…Finally, to evaluate GlcN effects on human skeletal muscle cells we used cultured human skeletal muscle cells that display several features of mature skeletal muscle and that have been previously used for studies of muscle metabolism [20]. In differentiated human muscle cells, GlcN induced a significant increase of both BIP/GRP78 and ATF6 mRNA levels (Fig.…”

Are cultured human myotubes far from home?

“…Studies performed in rodents have demonstrated that MyHC isoforms expressed in differentiated myotubes were identical to the MyHC expression pattern in the muscle fibers from which the satellite cells were isolated (Dusterhoft andPette, 1993, Rosenblatt, et al, 1996). In human muscle cell cultures it has been found that all myotubes in 8-day differentiated cell cultures were immunoreactive for fast MyHC, regardless of donor muscles having mixed MyHC expression in vivo (Gaster, et al, 2001) . In our hands, myotube cultures stained at day 8 after differentiation contained a noteworthy amount of slow muscle fibers as well (Fig.…”

Reduced Lipid Oxidation in Skeletal Muscle From Type 2 Diabetic Subjects May Be of Genetic Origin

“…First, satellite cultures express traits known from in vivo muscles (16 -20), and second, they allow the genetic and adaptive processes to be differentiated (21). Recently, we described the optimized conditions for satellite culture proliferation and differentiation (19), which have been shown to be suitable for studying the regulation of glycogen synthase activity and the differentiation between induced and primary defects in glucose metabolism in diabetic and normal myotubes (20,21). In the present study, we measured palmitic acid uptake, oxidation, and incorporation into complex cellular lipids in myotubes established from type 2 diabetic subjects and matched control subjects to clarify whether myotubes established from the diabetic subjects expressed inherited defects in fatty acid metabolism.…”

“…Cell culture. Cell cultures were established as previously described (19,20). In brief, muscle tissue was minced, washed, and dissociated for 60 min by three treatments with 0.05% trypsin-EDTA.…”