ABSTRACT
Myogenic precursors sense and dynamically respond to mechanical stimulation through complex integrin-mediated mechanotransduction, in which focal adhesion kinase (FAK) is a fundamental intracellular signaling mediator. When skeletal myoblasts are exposed to uniaxial cyclic tensile strain (UCTS), they display uniform alignment and an enhanced rate of differentiation. In this work, we explored the role of FAK activation by using C2C12 myoblasts that were grown on flexible culture plates and exposed to UCTS during the early differentiation phase. After 24 h, the cells oriented perpendicularly to the direction of strain and exhibited an enhanced differentiation profile. Next, the cells were exposed to a strain field that was either kept in the same direction or rotated 90°, in the presence or not of an FAK phosphorylation inhibitor. On reorientation of the strain field by 90°, the cells reassembled their focal adhesions and actin cytoskeleton to regain the perpendicular position with respect to the engaging stress. After blocking the FAK, however, the cells failed to respond to the reoriented strain field and their differentiation was abrogated. Interestingly, when the strain field remained in the same direction, the FAK inhibitor compromised the differentiation, even though there was no evident change in cell orientation. Our data indicate that during exposure to UCTS, the activation of FAK is necessary for the myoblasts to undergo alignment and enhanced differentiation.
Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/metabolism , Mechanotransduction, Cellular/physiology , Myoblasts/cytology , Myoblasts/metabolism , Animals , Blotting, Western , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line , Mechanotransduction, Cellular/genetics , Mice , Muscle Development/genetics , Muscle Development/physiology , Phosphorylation/genetics , Phosphorylation/physiology , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
BACKGROUND: Complex immunophenotypic repertoires defining discrete adipose-derived stem cell (ASC) subpopulations may hold a key toward identifying predictors of clinical utility. To this end, we sorted out of the freshly established ASCs four subpopulations (SPs) according to a specific pattern of co-expression of six surface markers, the CD34, CD73, CD90, CD105, CD146, and CD271, using polychromatic flow cytometry. METHOD: Using flow cytometry-associated cell sorting and analysis, gating parameters were set to select for a CD73+CD90+CD105+ phenotype plus one of the four following combinations, CD34-CD146-CD271- (SP1), CD34-CD146+CD271- (SP2), CD34+CD146+CD271- (SP3), and CD34-CD146+CD271+ (SP4). The SPs were expanded 700- to 1000-fold, and their surface repertoire, trilineage differentiation, and clonogenic potential, and the capacity to support wound healing were assayed. RESULTS: Upon culturing, the co-expression of major epitopes, the CD73, CD90, and CD105 was maintained, while regarding the minor markers, all SPs reverted to resemble the pre-sorted population with CD34-CD146-CD271- and CD34-CD146+CD271- representing the most prevalent combinations, followed by less frequent CD34+CD146-CD271- and CD34+CD146+CD271- variants. There was no difference in the efficiency of adipo-, osteo-, or chondrogenesis by cytochemistry and real-time RT-PCR or the CFU capacity between the individual SPs, however, the SP2CD73+90+105+34-146+271- outperformed others in terms of wound healing. CONCLUSIONS: Our study shows that ASCs upon culturing inherently maintain a stable distribution of immunophenotype variants, which may potentially disguise specific functional properties of particular downstream lines. Furthermore, the outlined approach suggests a paradigm whereby discrete subpopulations could be identified to provide for a therapeutically most relevant cell product.
Subject(s)
Adipocytes/cytology , Chondrocytes/cytology , Osteoblasts/cytology , Stem Cells/classification , Stem Cells/cytology , Adipocytes/metabolism , Adult , Antigens, CD/genetics , Antigens, CD/metabolism , Biological Assay , Cell Differentiation , Cell Lineage/genetics , Chondrocytes/metabolism , Flow Cytometry , Gene Expression , Humans , Immunophenotyping , Male , Osteoblasts/metabolism , Phenotype , Primary Cell Culture , Stem Cells/metabolism , Subcutaneous Fat, Abdominal/cytology , Subcutaneous Fat, Abdominal/metabolismABSTRACT
Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.