Proliferative potential of larval cells of the mussel Mytilus trossulus and their capacity to differentiate into myogenic cells in culture

Abstract

Cell differentiation and proliferation can be regulated by the extracellular matrix. To compare cell proliferation and myogenic differentiation in cultivated Mytilus larval cells on different substrates (collagen I and fibronectin), a double immunostaining with subsequent confocal microscopy were used for simultaneous detection of dividing and muscle cells. The proliferative activity was monitored using two markers, proliferating cell nuclear antigen (PCNA) and phospho-histone H3. The maximum number of mitotic (phosphohistone H3-positive) cells was observed after 4 h of cultivation (approximately 3%), but later, after 48 h, it decreased to 0.5%. Most of these cells formed small aggregates on all substrates tested. After 24 h of cultivation, the number of mitotic cells was approximately 5–7 times lower than that of S-phase (PCNA-positive) cells. The decrease in cell proliferation was accompanied by intensification of myogenic differentiation. First muscle cells were detected after 6 h of cultivation on fibronectin; numerous contracting muscle cells were observed after 24 h of cultivation. In contrast, the cells cultivated on collagen had mostly a rounded shape, did not spread, and showed a contracting activity only in rare cases. A small number of muscle cells with PCNA-positive nuclei were detected after 3-day-cultivation on fibronectin. We suggest that at early stages of cultivation, muscle precursor cells, or myoblasts, are able to synthesize DNA but lose this ability later.