Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells

Abstract

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.

The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model

“…suggest that MSC myogenic potential depends on the cell source. Authors have proved desmin protein expression in MSC derived from adipose tissue treated with myoblasts-conditioned medium, but not in bone marrow MSC [27]. Similarly, Belema-Bedada et al .…”

Engineering Approaches for Creating Skeletal Muscle

“…Embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) have all been shown to differentiate into myoblast-like cells and may be able to release beneficial cytokines to enhance wound healing. [27][28][29][30][31][32][33][34][35] These cell types are currently at the forefront of muscle tissue engineering.…”

“…33 More recently, MSCs have been shown to support muscle cell growth, and can differentiate and fuse to form multinucleated myofibers. 34 There is increasing evidence that MSCs are involved in the upkeep of various tissues, especially through the release of growth factors and regulation of the behavior of monopotent tissue stem cells. 35 MSCs are also known to play a role in modulating the immune response through various mechanisms including contact inhibition of T-cells and B-cells.…”

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

“…One interesting candidate marker to select for myogenic precursors is CD34, expressed in quiescent satellite cells and proliferating myogenic precursors (Beauchamp et al, 2000;Jankowski et al, 2002;Zammit et al, 2006). Additionally, factors involved in promoting the myogenic potential of stem cells are produced and secreted by muscle stem or progenitor cells that in turn enhance in vitro differentiation of stem cells into the myogenic lineage (Stern-Straeter et al, 2014).…”