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Isolated cardiac muscle cells grown in vitro have been studied with respect to their ability to contract spontaneously and maintain myofibrillar organisation during division. These cells do not round up to undergo mitosis; division is achieved by the cell pinching itself in two in a selected area. This adaptation minimises disturbance to cell attachment sites and to myofibrils running between them. We correlated this with the persistence of beating during division and the maintenance of myofibrils with intact Z bands, even in close proximity to the nucleus, through division in many cells. Cessation of beating and disorganisation of myofibrils are therefore not prerequisites for division of cardiac muscle cells, as reported previously.

A single, functional, mitotically quiescent chondrocyte may be induced to reenter the mitotic cyde, and produce a progeny of over 1011 cells. Sessile, adherent, polygonal cells deposit matrix, whereas amoeboid, dispersed, flattened fibroblastic cells do not. The prior synthetic history of a cell is of greater importance in determining whether the characteristic chondrogenic phenotype will be expressed, rather than growth in "permissive" or "nonpermissive" medium. Clonal conditions select for stem-like cells, some of whose progeny may become polygonal chondrocytes. The retention of the characteristic chondrogenic phenotype in vitro is favored by pruning the dedifferentiated chondrocytes which arise in these cultures. Dedifferentiated chondrocytes interfere with the deposition and synthesis of chondroitin sulfate by neighboring functional chondrocytes. Possible mechanisms are proposed to explain this type of cell-cell or cell exudate interference. If the progeny of a single, genetically programmed chondrocyte may or may not synthesize chondroitin sulfate, then extragenic sites in the cytoplasm or cell surface must influence the decision as to which cluster of "luxur" molecules the cell will synthesize.

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.

Cardiac muscle cells obtained by trypsinizing 5-day chick embryonic heart were cultured as single cells in separate culture dishes. Using this technique, problems of heterotypic cell interactions, “overgrowth” of one cell type, etc., are eliminated. Experiments performed on these single cell cultures show that the muscle cells in the embryonic chick hearts differ in morphology, including content of cross-striated myofibrils; in ability to synthesize DNA and undergo mitosis; and in frequency of contraction. Contracting cells containing cross-striated myofibrils undergo mitosis in vitro, giving rise to spontaneously beating daughter cells. These daughter cells contain cytoplasmic fibrils, which bind fluorescein-labeled antimyosin immediately after cytokinesis. Some cardiac muscle cells from 5-day heart do not divide in culture; the rest undergo 1–5 doublings. This preliminary investigation suggests that the new muscle cells formed during cardiac growth are derived from mitotically active “overtly” differentiated cardiac muscle cells.