Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.
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.
Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty Mannheim, University of Heidelberg, 68167 Mannheim, Germany.
Jens
J
Stern-Straeter
Gabriel Alejandro
GA
Bonaterra
Stephanie
S
Juritz
Richard
R
Birk
Ulrich Reinhart
UR
Goessler
Karen
K
Bieback
Peter
P
Bugert
Johannes
J
Schultz
Karl
K
Hörmann
Ralf
R
Kinscherf
Anne
A
Faber
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology* / Q000187:drug effects; D001374:Azacitidine / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry* / Q000494:pharmacology; D003893:Desmin / Q000235:genetics / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects*; D024510:Muscle Development / Q000187:drug effects*; D018482:Muscle, Skeletal / Q000166:cytology* / Q000187:drug effects; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism
D000199:Actins; D003470:Culture Media; D003893:Desmin; C497417:MYF5 protein, human; C488292:MYH8 protein, human; D051787:Myogenic Regulatory Factor 5; D018995:Myosin Heavy Chains; D001374:Azacitidine