MicroRNA-1 and MicroRNA-206 Improve Differentiation Potential of Human Satellite Cells: A Novel Approach for Tissue Engineering of Skeletal Muscle

Abstract

Innovative strategies based on regenerative medicine, in particular tissue engineering of skeletal muscle, are promising for treatment of patients with skeletal muscle damage. However, the efficiency of satellite cell differentiation in vitro is suboptimal. MicroRNAs are involved in the regulation of cell proliferation and differentiation. We hypothesized that transient overexpression of microRNA-1 or microRNA-206 enhances the differentiation potential of human satellite cells by downregulation quiescent satellite cell regulators, thereby increasing myogenic regulator factors. To investigate this, we isolated and cultured human satellite cells from muscle biopsies. First, through immunofluorescent analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we showed that in satellite cell cultures, low Pax7 expression is related to high MyoD expression on differentiation, and, subsequently, more extensive sarcomere formation, that is, muscle differentiation, was detected. Second, using qRT-PCR, we showed that microRNA-1 and microRNA-206 are robustly induced in differentiating satellite cells. Finally, a gain-of-function approach was used to investigate microRNA-1 and microRNA-206 potential in human satellite cells to improve differentiation potential. As a proof of concept, this was also investigated in a three-dimensional bioartificial muscle construct. After transfection with microRNA-1, the number of Pax7 expressing cells decreased compared with the microRNA-scrambled control. In differentiated satellite cell cultures transfected with either microRNA-1 or microRNA-206, the number of MyoD expressing cells increased, and α-sarcomeric actin and myosin expression increased compared with microRNA-scrambled control cultures. In addition, in a three-dimensional bioartificial muscle construct, an increase in MyoD expression occurred. Therefore, we conclude that microRNA-1 and microRNA-206 can improve human satellite cell differentiation. It represents a potential novel approach for tissue engineering of human skeletal muscle for the benefit of patients with facial paralysis.

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

“…The model as described here has been used already in studies on pressure ulcer damage while using a cell line 15 . For regenerative medicine applications, the translation to human satellite cells has recently been made 6,16 . In addition, as an alternative source for meat consumption the technique has great potential 1 .…”

“…Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4 , neonatal muscle derived progenitor cells 5 , cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7 . Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture.…”

Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro

“…While C2C12 cells have provided invaluable information about key mechanistic steps in differentiation (31), extensive in vitro cultivation may lead to deviations from normal biological processes that are important for differentiation of myoblasts in a normal in vivo environment. Less is known about the dynamics of key differentiation factors in primary human myoblasts and how culture conditions affect the changes in these factors (3).In vitro, rodent myoblast differentiation to myotubes is influenced by many stimuli, including growth factors, substrate composition and mechanical properties, mechanical and electrical stimulation, and addition of microRNAs (16,23,26,28). Mechanical conditioning, or stretch, is an important physiological stimulus that has been applied to skeletal myofiber cultures to mimic in vivo exercise (4,23,26,34).…”

“…In vitro, rodent myoblast differentiation to myotubes is influenced by many stimuli, including growth factors, substrate composition and mechanical properties, mechanical and electrical stimulation, and addition of microRNAs (16,23,26,28). Mechanical conditioning, or stretch, is an important physiological stimulus that has been applied to skeletal myofiber cultures to mimic in vivo exercise (4,23,26,34).…”

miRNA Control of Tissue Repair and Regeneration

“…106 Human satellite cells, important in the tissue engineering of the skeletal muscle, may be treated with miR-1 and miR-206 to improve their differentiation potential. 107 Biomaterial scaffolds play a key role in scar formation associated with wound healing. These ECM-derived scaffolds, in addition to serving as physical support to promote tissue organization, resist aggressive wound contraction 108 and scar tissue formation.…”